Collagen fibrillar construction

ABSTRACT

Methods and compositions are described for organizing collagen into fibrillar networks, e.g, short and long-range organization. Collagen produced by the disclosed methods can be used for tissue engineering.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/901,286, filed Oct. 8, 2010, which is a continuation of InternationalApplication No. PCT/US2009/040364, filed Apr. 13, 2009, which claims thebenefit of U.S. Provisional Application No. 61/044,103, filed Apr. 11,2008, U.S. Provisional Application No. 61/045,439, filed Apr. 16, 2008,and U.S. Provisional Application No. 61/060,644, filed Jun. 11, 2008,the contents of all of which are hereby incorporated in their entiretyherein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with government support under Grant No.5R21AR053551-02, awarded by The National Institutes of Health. TheUnited States government has certain rights in this invention.

FIELD OF THE INVENTION

The invention is in the fields of tissue engineering and medicine.

BACKGROUND OF THE INVENTION

Though significant effort has been expended to “tissue-engineer” naturalgrafts for the replacement of damaged or diseased load-bearingextracellular matrices (ECMs) such as ligament, tendon and cornea, noclinically viable constructs have been produced. One of the mostdifficult problems associated with engineering connective tissue (whichtypically begins with weak, self-assembled collagen-based scaffolds) isthe achievement of adequate mechanical strength. The inability of suchconstructs to bear in vivo or near in vivo loads at the time ofimplantation has led to the development of methods which employ complexdegradable high-strength scaffolds (e.g., silk) onto which fibroblasticcells are seeded. Once seeded, the fibroblasts in the matrix arestimulated (chemically and/or mechanically) to replace the resorbingscaffold material with a natural collagen-based matrix. This approach ishampered by uncertainties in the kinetics of the degradation andreplacement process as well as the usual problems associated with theuse of non-native biomaterials.

Collagen molecules comprise an epigenetically adaptable load-bearingmatrix that can be culled by enzymatic degradation or reinforced byincorporation of monomers (longitudinally and in the radial direction).The generic ability of collagen-based matrices to add and removemonomers from fibrils is a remarkable feature of native load-bearingECM, as are its mechanical properties (due to collagen's high tensilemechanical strength). Such tissue can be characterized by its highlyanisotropic and ordered collagen fibrillar organization (cornea (Hay etal., Monogr. Dev. Biol. 1:1-144 (1969)), ligament and tendon (Provenzanoet al., Matrix Biol. 25:71-84 (2006)), and annulus fibrosus (Marchini etal., Basic Appl. Histochem. 23:137-148 (1979)). Unfortunately, collagenhas limited regenerative ability following injury (Fini et al., Cornea24:S2-S11 (2005); Frank, Musculoskelet. Neuronal Interact. 4:199-201(2004); Lotz et al., Neurosurg. Clin. N. Am. 16:657-663 (2005)).

Fibrillar collagen is the principal load-bearing molecule in vertebrateanimals and the most abundant protein in vertebrates. The untimelyfailure of collagenous load-bearing tissues (which are often refractoryto self-repair) due to degeneration, acute injury or collagen-relateddisease affects hundreds of millions of people world-wide and often canhave a devastating impact on the quality of life of the individual ifleft untreated. In the industrialized world, degenerative disease ofcollagen-based tissue has a high prevalence. In the US, 32 millionpeople over the age of 20 have frequent lower back pain; the majority ofthese cases are likely the result of intervertebral disc degeneration.The cost of indirect and direct medical care for herniated disks wasestimated to be $1.6B in 1995. Osteoarthritis, a degenerative cartilagepathology of unknown etiology, affects over 20 million US adults and issecond only to chronic heart disease as the reason for long-termdisability payment requests. Acute injury to connective tissue alsocontributes significantly to the loss of load-bearing tissue function.There are 200,000 anterior cruciate ligament (ACL) injuries and between60,000 and 95,000 ACL reconstructions are performed each year in the USto restore mechanical function.

There also are a constellation of collagen-related diseases due togenetic mutations which include Ehlers-Danlos syndrome, Bethlemmyopathy, Alport syndrome, Knobloch syndrome, osteoporosis (some cases),osteogenesis imperfecta, arterial aneurysm and rheumatoid arthritis(autoimmune).

Very little is known about the mechanisms which govern the organizationand morphology of collagen during synthesis by fibroblasts in vivo, forit is the loss of or damage to organized collagen that is oftenirreparable. The majority of tissue engineers have opted to investigatesynthetic biomaterials and have, with few exceptions, treated collagenas a “degradable” cell transport vehicle. There are more than 10 millioncases of corneal blindness (caused by both injury and disease), the vastmajority of which could benefit from suitable corneal replacement. Inthe US, 33,000 corneal transplants are performed each year in the US;however, recipients will be subject to a looming graft material shortageinduced by the extensive use of LASIK corrective surgery, which renderscorneas unsuitable for donation.

SUMMARY OF THE DISCLOSURE

The disclosure is based, at least in part, on the discovery thatcollagen monomers can be organized into long-range fibrillar networks byconfining solutions of concentrated collagen monomers within templates.Accordingly, in one aspect, the disclosure features a method ofproducing an organized array of collagen fibrils. The method includescontacting a template with a still solution comprising collagen monomersin liquid crystalline phase; and neutralizing the solution in contactwith the template, thereby inducing the assembly of the collagenmonomers into an organized array of collagen fibrils.

In some embodiments, the collagen monomers comprise a nematic phase. Inother embodiments, the collagen monomers comprise a smectic phase. Inyet other embodiments, the collagen monomers comprise a cholestericphase.

In certain embodiments, the solution comprises about 30 mg/ml to about1000 mg/ml collagen monomers, about 30 mg/ml to about 500 mg/ml, about40 mg/ml to about 400 mg/ml, about 50 mg/ml to about 300 mg/ml, about 60mg/ml to about 200 mg/ml, about 70 mg/ml to about 150 mg/ml, about 80mg/ml to about 125 mg/ml, about 90 mg/ml to about 120 mg/ml, or about100 mg/ml collagen monomers.

In some embodiments, the method includes neutralizing the solution byadjusting the solution to a pH of about 5 to about 10, about 5.5 toabout 9.5, about 6 to about 9, about 6.5 to about 8.5, or about 6.5 toabout 8.

In other embodiments, the method includes neutralizing the solution incontact with the template at about 10° C. to about 39° C., at about 10°C. to about 35° C., about 15° C. to about 30° C., or about 20° C. toabout 25° C.

In certain embodiments, the method further comprises applying anelectric charge to the template.

In particular embodiments, the template comprises one or more guidancestructures. In specific embodiments, the one or more guidance structuresare one or more internal guidance structures and the template is placedin a stationary position within the solution. In some embodiments, theguidance structures comprise a surface having a pattern of hydrophobicand hydrophilic stripes.

In other embodiments, the one or more internal guidance structurescomprise a high aspect ratio geometry. In particular embodiments, theone or more internal guidance structures comprise a minor length scaleof between about 14 nm and about 20 μm, for example, between about 20 nmand about 15 μm, between about 25 nm and about 10 μm, between about 30nm and about 5 μm, between about 40 nm and about 100 nm, between about50 nm and about 90 nm, between about 60 nm and about 80 nm, or about 70nm.

In some embodiments, one or more of the internal guidance structurescomprise a biodegradable material. In certain embodiments, thebiodegradable material is silk, PLGA, or a PLA-type material (such asPDLA, PLLA, or PDLLA).

In yet other embodiments, the template comprises a plurality of externalguidance structures. In some embodiments, the external guidancestructures have an interstructure distance of about 2 μm to about 200μm, for example about 4 μm to about 175 μm, about 8 μm to about 150 μm,about 10 μm to about 125 μm, about 20 μm to about 100 μm, about 30 μm toabout 90 μm, about 40 μm to about 80 μm, or about 50 μm to about 70 μm.In some embodiments, the template comprises one or more internalguidance structures and one or more external guidance structures.

In certain embodiments, the template comprises a cylindrical tube, twoconcentric cylindrical tubes, or two concentric hemispheres. In someembodiments, the template comprises a cylindrical tube having an innerdiameter of about 100 μm to about 1 mm, for example about 125 μm toabout 900 μm, about 150 μm to about 800 μm, about 175 μm to about 700μm, about 200 μm to about 600 μm, about 300 μm to about 500 μm, or about400 μm to about 450 μm. In other embodiments, the template comprises twoconcentric cylinders with a gap width of about 2 μm to about 4 mm, forexample, about 4 μm to about 2 mm, about 8 μm to about 1 mm, about 10 μmto about 900 μm, about 20 μm to about 800 μm, about 30 μm to about 700μm, about 40 μm to about 600 μm, about 50 μm to about 500 μm, about 100μm to about 400 μm, or about 200 μm to about 300 μm. In yet otherembodiments, the template comprises two concentric hemispheres with agap width of about 2 μm to about 4 mm, for example, about 4 μm to about2 mm, about 8 μm to about 1 mm, about 10 μm to about 900 μm, about 20 μmto about 800 μm, about 30 μm to about 700 μm, about 40 μm to about 600μm, about 50 μm to about 500 μm, about 100 μm to about 400 μm, or about200 μm to about 300 μm.

In some embodiments, the template comprises a scaffold that mimics acornea, a ligament, a tendon, a meniscus, an intervertebral disk, orarticular cartilage.

In certain embodiments, the collagen monomers are selected from thegroup consisting of Type I collagen monomers, Type II collagen monomers,Type III collagen monomers, Type V collagen monomers, Type XI collagenmonomers, an MMP-resistant mutant thereof, and combinations thereof. Inother embodiments, the collagen monomers are selected from the groupconsisting of atelo-collagen monomers, tropocollagen monomers,procollagen monomers, and combinations thereof.

In particular embodiments, the solution comprises a buffer or saltselected from the group of CaCl₂, NaOH, NaCl, Na₂HPO₄, NaHCO₃, Hepes,PBS, Trizma base, Tris-Hcl, cell culture media, and combinationsthereof.

In yet other embodiments, the solution comprises one or moreco-nonsolvency agents. In certain embodiments, the co-nonsolvency agentis polyethylene glycol, hyaluronic acid, a glycosaminoglycan, aproteoglycan, or a combination thereof. In some embodiments, theglycosaminoglycan is chondroitin sulfate, hyaluronic acid, heparin,heparin sulfate, keratin sulfate, or dermatan sulfate.

In other embodiments, the solution further comprises a collagen bindingagent. In some embodiments, the collagen binding agent is aproteoglycan, a glycoprotein, a collagen-binding portion thereof, or acombination thereof. In certain embodiments, the proteoglycan islumican, decorin, biglycan, perlecan, versican, fibromodulin, aggrecan,sydecan or a combination thereof. In other embodiments, the glycoproteinis fibronectin, laminin, osteonectin, or a combination thereof.

In yet other embodiments, the organized array of collagen fibrils isabout 100 μm to about 30 cm in length, for example, about 200 μm toabout 20 cm, about 400 μm to about 10 cm, about 500 μm to about 5 cm,about 750 μm to about 1 cm, about 1 mm to about 500 mm, about 10 mm toabout 400 mm, about 50 mm to about 300 mm, about 100 mm to about 200 mm,or about 100 mm to about 150 mm.

In some embodiments, the organized array of collagen fibrils comprisesD-banded collagen fibrils.

In certain embodiments, the method further comprises contacting thecollagen monomers in the organized array of collagen fibrils with acrosslinking agent. In some embodiments, the crosslinking agent isformaldehyde, hexamethylene diisocyanate, glutaraldehyde, a polyepoxycompound, gamma irradiation, ultraviolet irradiation with riboflavin,transglutaminase, acyl azidesglycidyl ethers, diisocyanates,hexamethylenediisocyanate, bis-epoxide, carbodiimide,dimethylsuberimidate, nordihydroguaiaretic acid, lysyl oxidase, or acombination thereof.

In other embodiments, the method further comprises modulating thesurface energy of the guidance structures. In some embodiments, thesurface energy is modulated by plasma cleaning, silanization, orhydrophobic/hydrophilic bonding.

In some embodiments, the method further comprises organizing the arrayof collagen fibrils into a structure mimetic of a load-bearing tissue bycontacting the array with a secondary template. In certain embodiments,the secondary template comprises cells, such as primary fibroblasts orstem cells, and forms a load-bearing tissue of a given morphology.

In another aspect, the invention features a method of producing anorganized array of collagen fibrils. The method comprises contacting atemplate with a solution comprising collagen monomers to form collagenfibrils in the solution; applying tension to a plurality of collagenfibrils in the solution, where the tension produces a strain of about 1%to about 20%; and contacting the collagen fibrils with a collagen lyticprotease; thereby producing an organized array of collagen fibrils fromthe plurality of collagen fibrils by selective removal of fibrils withlower strain. In some embodiments, applying tension to the plurality ofcollagen fibrils protects the plurality of collagen fibrils fromenzymatic degradation by the collagen lytic protease.

In some embodiments, the collagen lytic protease is a bacterialcollagenase, a matrix metalloproteinase (MMP), cathepsin K, or abiologically active fragment thereof.

In some embodiments, the method further comprises neutralizing thesolution in contact with the template. In certain embodiments, themethod includes neutralizing the solution by adjusting the solution to apH of about 5 to about 10, for example, about 5.5 to about 9.5, about 6to about 9, about 6.5 to about 8.5, or about 6.5 to about 8. In otherembodiments, the method includes neutralizing the solution in contactwith the template at about 10° C. to about 39° C., for example, at about10° C. to about 35° C., about 15° C. to about 30° C., or about 20° C. toabout 25° C.

In certain embodiments, the tension produces a fibril strain of about 1%to about 20%, for example, about 1% to about 10%, about 1% to about 9%,about 2% to about 8%, about 3% to about 7%, or about 4% to about 6%. Inother embodiments, the tension produces a strain of about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, orabout 10%.

In some embodiments, the tension is applied to the fibrils in a constantmanner. In other embodiments, the tension is applied to the fibrils inan oscillatory manner. In particular examples, the tension produces anoscillatory strain of about 1% to about 10% amplitude on the fibrils. Incertain embodiments, the tension produces an oscillatory strain at afrequency of about 0.1 Hz to about 5 Hz, for example, about 0.2 Hz toabout 4 Hz, about 0.3 Hz to about 3 Hz, or about 0.5 Hz to about 2 Hz.

In certain embodiments, the tension is applied to both ends of theplurality of collagen fibrils.

In other embodiments, the method further comprises adding supplementalcollagen monomers to the solution after tension is applied. In someembodiments, the collagen lytic protease and the supplemental collagenmonomers are added simultaneously to the solution. In other embodiments,the collagen lytic protease and the supplemental collagen monomers areadded sequentially to the solution. In yet other embodiments, thecollagen lytic protease and the supplemental collagen are added morethan once to the solution.

In some embodiments, the method further comprises organizing the arrayof collagen fibrils into a tissue.

In another aspect, the invention features an organized array of collagenfibrils produced by any of the methods described herein.

In another aspect, the invention features a method of directing theassembly of collagen fibrils. The method comprises contacting a templatewith a still solution comprising collagen monomers in liquid crystallinephase, the template comprising a plurality of guidance structures; andneutralizing the solution in contact with the template; whereincontacting the template directs the assembly of the collagen monomers ina pattern or direction defined by the guidance structures.

The following figures are presented for the purpose of illustrationonly, and are not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a representation of a TEM micrograph of organized,alternating collagen lamellae deposited by human primary cornealfibroblasts in vitro at 28 days where bar is 2μ. Fibril directionchanges in the human culture system are indicated by the black arrows;Col=collagen; fl=flocculent material.

FIG. 1B is a representation of a TEM micrograph of organized,alternating collagen lamellae deposited by human primary cornealfibroblasts during the development of a non-human primate (macacque)cornea in vivo at 60 days. Fibril direction changes in the human culturesystem are indicated by the black arrows; Col=collagen; fl=flocculentmaterial.

FIG. 2A is a representation of a phase contrast image of 2 week old invitro primary human corneal stromal cell (PHCSC)-derived collagenconstructs, where bar is 50μ.

FIG. 2B is a representation of an optical thick section of 8 week oldPHCSC-derived, stratified collagen construct comprising PHCSCs with“orthogonal” orientation changes (white and black arrowheads) and layerswhere aligned collagen lamellae are typically observed (black arrows),and the inset depicts an sTEM of single PHCSC in cross-sectionsurrounded by collagen fibrils aligned with cell long axis. Thisdemonstrates internal templating by the resident cells.

FIG. 2C is a representation of an sTEM of cell-synthesized collagenousmatrix comprising alternating layers of small diameter collagen fibrils,where bar is 500 nm.

FIG. 2D is a representation of an optical thick section of a lowconcentration (LC) liquid-crystal-collagen-derived de novo construct,where five morphologically distinct layers (at low magnification lightmicroscopy) are clearly visible in this 100 micron thick cross-section(white arrows indicate individual layers).

FIG. 3A is a representation of a DIC optical micrograph of de novocollagen constructs from low concentration of collagen monomers.

FIG. 3B is a representation of a DIC optical micrograph of de novocollagen constructs from high concentration of collagen monomers.

FIG. 3C is a representation of a DIC optical micrograph extracted fromz-scans of de novo collagen constructs from low concentration ofcollagen monomers. The matrix alignment angle changes 30° over a depthof 30μ. The bar is 20μ.

FIG. 3D is a representation of a DIC optical micrograph extracted fromz-scans of PHCSC-derived collagenous matrix alignment. The matrixalignment angle changes 90° over a depth of 7μ. The bar is 20μ.

FIG. 3E is a representation of a DIC optical micrograph extracted fromz-scans of de novo collagen constructs from low concentration ofcollagen monomers. The matrix alignment angle changes 30° over a depthof 30μ. The bar is 20μ.

FIG. 3F is a representation of a DIC optical micrograph extracted fromz-scans of PHCSC-derived collagenous matrix alignment. The matrixalignment angle changes 90° over a depth of 7μ. The bar is 20μ.

FIG. 4A is a representation of a cross-section low magnification sTEMmicrograph of human corneal lamellae.

FIG. 4B is a representation of a cross-section low magnification sTEMmicrograph of lamellar structures spontaneously formed in de novoconstruct at low concentration of collagen. Black arrows indicate thewidth of a lamella, and white arrow indicates the direction of fibrilalignment in a lamellar plate, where bar is 2μ.

FIG. 4C is a representation of a cross-section low magnification sTEMmicrograph of lamellar structures formed in de novo constructs at highcollagen concentration, where bar is 5μ.

FIG. 4D is a representation of a cross-section low magnification sTEMmicrograph of lamellar structures formed in de novo constructs at highcollagen concentration, where bar is 2μ.

FIG. 5A is a representation of a sTEM micrograph of PHCSC cells in a 4week old construct co-aligned with collagen fibrils, where collagenfibrillar arrays change direction at the white line; below the whiteline, collagen fibrils and cells are in cross-section (CS); and abovethe line cells and fibrils are in oblique section (OS), where blackdouble arrow indicates the distance over which cell orientation could beinfluencing the fibril orientation, and bar is 4μ.

FIG. 5B is a representation of a DIC micrograph of a glass microcylinderembedded in fibrils precipitated from liquid crystalline collagenmonomers, where black double arrow indicates the distance over which theglass “guides” collagen; white double arrow indicates direction offibrils, and bar is 20μ.

FIG. 6A is a representation of a SEM micrograph of fracturedcross-section of collagen in high-concentration de novo construct, andinsets depict a clear change of direction in highly-aligned collagenfibrils in lamellae, where lower black arrow indicates fibrils that arecut in cross-section that are densely packed and that appear to be“solid,” and bar is 10μ.

FIG. 6B is a representation of a low magnification sTEM micrograph of alarge uniform area in cross-section showing the high-density anduniformity of the fibrillar array, where bar is 2μ.

FIG. 6C is a representation of a high magnification sTEM micrograph ofcollagen in cross-section showing the presence of individual fibrils,where bar is 500 nm.

FIG. 7A is a representation of a sTEM micrograph of collagen from lowconcentration collagen monomers, where inset is a higher magnification,and bar is 100 nm.

FIG. 7B is a representation of a sTEM micrograph of collagen from highconcentration collagen monomers, where inset is a higher magnification,and bar is 100 nm.

FIG. 7C is a representation of a sTEM micrograph of collagen from highconcentration collagen monomers, and bar is 100 nm.

FIG. 7D is a representation of a sTEM micrograph of PHCSC-derivedcollagen, where bar is 100 nm.

FIG. 8 is a diagrammatic representation of channels etched into acoverslip.

FIG. 9A is a diagrammatic representation of a single external template.

FIG. 9B is a schematic representation of multiple templates.

FIG. 9C is a schematic representation of perpendicular externaltemplates.

FIG. 10A is a schematic representation of single internal template.

FIG. 10B is a schematic representation of multiple internal templates.

FIG. 10C is a schematic representation of perpendicular internaltemplates.

FIG. 11 is a diagrammatic representation of concentric glass cylindersto make concentric lamellae.

FIG. 12 is a diagrammatic representation of concentric hemispheres tomake cornea-like collagen.

FIG. 13A is a graphic representation of % strain versus time curves fornative tissue strip in a uniaxial loading bioreactor (load control).

FIG. 13B is a graphic representation of estimates of effectiveloaded-area loss as a function of time for native tissue strip in auniaxial loading bioreactor.

FIG. 14A is a representation of a photograph of a cartilage plug with acapillary tube needed inserted.

FIG. 14B is a representation of a photograph of a dehydrated cartilageplug loaded with MMP-13 solution.

FIG. 14C is a representation of a photograph of a rehydrated cartilageplug ready to be warmed and mechanically examined.

FIG. 15 is a graphic representation comparing the net loss (percentagedrop) in load versus time due to collagenase at 10% and 20% strain.

FIG. 16A is a diagrammatic representation of a system to carry outdegradation of strained collagen in micro chamber while observing theprocess using DIC microscopy.

FIG. 16B is an inset schematically depicting the micropipette positionand collagen gel in strained and unstrained form inside the microchamber.

FIG. 17 is a graphic representation of the quantification of edge lossagainst digestion time ΔT in control sample (no strain).

FIG. 18A is a representation of a DIC image of preferential,strain-directed degradation of a reconstituted collagen gel by bacterialcollagenase at time 00:00:30.

FIG. 18B is a representation of a DIC image of preferential,strain-directed degradation of a reconstituted collagen gel by bacterialcollagenase at time 00:52:00.

FIG. 18C is a representation of a DIC image of preferential,strain-directed degradation of a reconstituted collagen gel by bacterialcollagenase at time 01:00:00.

FIG. 19 is a graphic representation of the quantification of edge lossagainst digestion time ΔT.

FIG. 20A is a representation of a DIC image of degradation of areconstituted collagen gel by MMP-8 at time 00:01:00.

FIG. 20B is a representation of a DIC image of degradation of areconstituted collagen gel by MMP-8 at time 01:53:00.

FIG. 20C is a representation of a DIC image of degradation of areconstituted collagen gel by MMP-8 at time 04:12:30.

FIG. 21A is a representation of a DIC image of growth of a loadedcluster of reconstituted collagen fibrils at time 00:00:00.

FIG. 21B is a representation of a DIC image of growth of a loadedcluster of reconstituted collagen fibrils at time 00:02:01.

FIG. 21C is a representation of a DIC image of growth of a loadedcluster of reconstituted collagen fibrils at time 00:05:32.

FIG. 21D is a representation of a DIC image of growth of a loadedcluster of reconstituted collagen fibrils at time 00:10:20.

FIG. 22 is a graphic representation of strains at various times forcorneal stromal collagen degradation by BC based on a model.

FIG. 23 is a diagrammatic representation of a device for measuring thekinetics of collagen monomer polymerization, where (a) indicates FITClabeled collagen monomers polymerizing onto stretched fibrils, (b)represents stretched collagen fibrils, (c) represents micropipettes(programmed for inducing static/cyclic strain), and (d) depicts collagenfibrils formed in unloaded matrix.

FIG. 24 is a diagrammatic representation of a chamber modified tofacilitate collagenous tissue growth.

FIG. 25A is a representation of a Solidworks rendered image of auniaxial loading system for growing construct strips under load.

FIG. 25B is a representation of a Solidworks image of a tangentialloading chamber for growing whole constructs under tangential load.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein,including GenBank database sequences, are incorporated by reference intheir entirety. In case of conflict, the present specification,including definitions, will control. In addition, the materials,methods, and examples are illustrative only and not intended to belimiting.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

DEFINITIONS

As used herein, a “template” is a three-dimensional structure orsubstrate that controls collagen fibril organization. A template createsa zone of local influence within a solution of collagen. A templatecomprises one or a plurality of guidance structures.

As used herein, a “guidance structure” is a structure with a high aspectratio with a minor length scale of between about 14 nm and about 20 μm.The guidance structure is defined by the operative length scale of acollagen monomer.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. The term “about” is usedherein to mean approximately, in the region of, roughly, or around. Whenthe term “about” is used in conjunction with a numerical range, itmodifies that range by extending the boundaries above and below thenumerical values set forth. In general, the term “about” is used hereinto modify a numerical value above and below the stated value by avariance of 20%. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another embodiment. It will be further understood that theendpoints of each of the ranges are significant both in relation to theother endpoint, and independently of the other endpoint.

As used herein, “interstructure distance” means the distance between theouter surfaces of two adjacent guidance structures.

As used herein, “alignment” in reference to collagen fibrils means thatmost of the fibrils in the same radial plane in a tube wall run roughlyparallel to each other. It is not meant that every fibril must beparallel to every other fibril in the plane, but that a generalalignment pattern must be discernable.

As used herein, a “fibril” is an association of several collagenmonomers into a structure that appears fibrous with suitablemagnification.

As used herein, “collagen” means a protein component of an extracellularmatrix having a tertiary structure that includes polypeptide chainsintertwining to form a collagen triple helix or having a characteristicamino acid composition comprising Gly-X-Y repeat units, or a fragmentthereof. Collagens can be any collagen known in the art (e.g., one ofcollagen Type 1-29).

General

The methods described herein are based, at least in part, on thediscovery that collagen and its complement matrix and enzymes form thebasis of a “smart” engineering material on multiple levels, includingthe ability to self-organize over both short and long length scales.Aligned layers of collagen fibrils can be produced by precipitatingfibrils from liquid crystalline collagen. The precipitated monomers canproduce naturally load bearing fibrillar structures. As the thickness ofthe sample can be controlled, the methods described herein can be usedin artificial tissue engineering, e.g, to produce both the short andlong-range organization and morphology of collagen in highly-anisotropicnative tissues such as tendon, ligament, bone, annulus fibrosus andcornea. Further, the absence of toxic material in the methods describedherein can result in a biocompatible construct. Finally, the collagenproduced by the methods described herein has much higher mechanicalstrength compared to regular collagen gels.

Collagen

The methods described herein can be used to produce organized collagen,e.g, to engineer tissues. Collagen is the most abundant protein in theextracellular matrix (ECM) of vertebrates and is the most commonstructural molecule in tensile load-bearing applications.

More than 29 different collagenous sequences are known. Fibrillarcollagens (e.g, Types I, II, III, V and XI) are the principal structuralcomponent in load-bearing extracellular matrix (ECM), which provides anetwork for cells to interact and form three dimensional, multi-cellularorganisms. Collagen possesses a linear-helical structure comprisingthree left-handed helical alpha chains whose complementary amino acidsequence results in the formation of a right-handed supramoleculartriple helix. Collagen contains the repetitive sequence amino acidsequence Gly-X-Y, where X and Y are usually proline and hydroxyproline,respectively.

As described herein, collagen is not a passively manipulated element,but rather a principal component in a cooperative engineering materialsystem, a system that significantly enhances the ability of fibroblasticcells to produce and optimize load-bearing tissue.

Any known collagen can be used in the methods described herein and canbe isolated or derived from a natural source, manufactured biochemicallyor synthetically, produced through genetic engineering, or producedthrough any other means or combinations thereof. In addition, collagenis commercially available (e.g, from Inamed Biomaterials, Fremont,Calif.; and FibroGen, Inc., San Francisco, Calif.). Natural sourcesinclude, but are not limited to, collagens produced by or containedwithin the tissue of living organisms (e.g, cows, pigs, birds, fish,rabbits, sheep, mice, rats, and humans). Further, natural collagen canbe obtained from, for example, tendons, bones, cartilage, skin, or anyother organ by any known extraction method. Exemplary sources includerat tail tendon and calf skin.

Some collagens that are useful in the methods described herein include,but are not limited to, collagen Types I, II, III, IV, V, VI, VII, VIII,IX, X, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, and XIX. Syntheticcollagen can include collagen produced by any artificial means, andnumerous methods for producing collagens and other proteins known in theart can be used. For example, synthetic collagen can be prepared usingspecific sequences, such as specific amino acids that are the same orthat differ from natural collagen. Engineered collagen can be producedby any method known in the art including, for example, polypeptidesynthesis.

Structural Arrangement of Collagen in Native Load-Bearing ECMs

The mechanics of native collagenous matrices are based on the nanoscaleorganization of the collagen fibrils. With the exception of bone (due toits unique remodeling mechanism comprising Haversion systems), thestructure of load-bearing tissues is “set” during development and doesnot appreciably change in the general long-range organization in theadult. There are principally two organizational regimes depicted for thecollagen fibrils: 1) uniaxial prismatic cylinders, and 2) uniaxialsheets or “lamellae”. In the case of the latter, 3-dimensionalstructures are “built-up” by successive layering of these lamellae instacks where the angle between lamellae is changed. The stacks can beformed in concentric cylinders (such as in lamellar and osteonal bone,annulus fibrosus) or nested hemispheres (such as in cornea). Thesestructures are actually 2-D (plus) and are naturally optimized forbearing tension in the plane of the lamellae (such as in cornea) or forresisting torsion (such as in annulus fibrosus).

Methods of Organizing Collagen

Concentrating and Precipitating Collagen

In some instances, the methods described herein include confining asolution of collagen monomers within a template having a definedconfinement geometry (e.g, having defined external guidance structures).The solution can include any type of collagen monomers. However, certainmethods utilize the same type of collagen as the collagen that mainlyconstitutes a particular tissue of interest. For example, for skin,bones, and tendons, Type I collagen can be used; for cartilage, Type IIcan be used; and for skin and muscles, Type III can be used.

The collagen in solution can be in a liquid crystalline phase, e.g., innematic, smectic, or cholesteric phase. In certain instances, theconcentration of collagen monomers in the solution is between about 30mg/ml and about 500 mg/ml. In other instances, the collagen solutionincludes a buffer. Examples of buffers include, without limitation,CaCl₂, NaOH, NaCl, Na₂HPO₄, NaHCO₃, Hepes, PBS, Tris, cell culturemedia, and combinations thereof.

By confining the solution of collagen monomers within external guidancestructures, the collagen monomers are induced to precipitate and to formcollagen arrays having a desired tissue architecture. In one exemplarymethod, collagen monomers are concentrated to about 100 mg/ml and areconfined between featureless planar glass coverslips separated by about40μ, leading to fibril precipitation from the solution with ahigh-degree of alignment in planes parallel to the coverslips. In thissituation, the coverslips provide external guidance structures thatinduce the alignment of the fibrils. Further, the collagen fibrils canform layers in which the orientation of the alignment of the fibrils canchange direction, forming a natural load-bearing structure similar tonative collagen organization found in cornea, bone, blood vessel intimaor adventitia, and annulus fibrosus. Thus, the concentration andconfinement of collagen in axially symmetric geometries can result inthe formation of structures similar to any collagenous tissue, such asligament or tendon.

In other methods, the local organization of collagen is controlled byusing internal templates (e.g., internal guidance structures). While notwishing to be bound by theory, it is believed that such internaltemplates mimic embedded fibroblasts (or fibroblast filipodia) toinfluence the local organization of collagen fibrils. In certaininstances, the internal template is made of a biodegradable polymer.Nonlimiting examples of biodegradable polymers include silk,poly(lactide), poly(glycolic acid), poly(lactide-co-glycolide),poly(caprolactone), polycarbonates, polyamides, polyanhydrides,polyamino acids, polyortho esters, polyacetals, polycyanoacrylates, anddegradable polyurethanes.

In one exemplary method, an internal template is made of fine degradablefilaments that are woven into a sparse scaffold. The scaffold can thenbe immersed in a solution of concentrated collagen monomers, whichinduces the alignment of the precipitating collagen to follow theinternal template. The spacing and size of the internal template (e.g,biodegradable filaments) can be arranged to result in a particularcollagen fibrillar organization of interest. Thus, the use of internalguidance structures allows internal control over the organization ofcollagen, such as solutions of collagen monomers confined within aparticular geometry (e.g, within external guidance structures).

Modifying External and Internal Templates

The external and internal templates (e.g., external and internalguidance structures) can be modified to influence the collagen fibrillarorganization. For example, using known methods, the surfaces of theexternal and internal templates can be plasma cleaned, patterned, orfunctionalized in other ways to control the local organization of theinterfacing fibrils to produce collagen arrays. In particular methods,the surfaces of the external and/or internal templates are silanated orcarbodiimidated using known methods.

In some instances, the surface charges of collagen molecules can be usedto direct the process of collagen assembly by applying an electriccharge to one or more surfaces of the external and/or internaltemplates. In particular methods, collagen molecules can be confinedbetween two metallic plates containing an electrical field to direct theassembly of collagen. In other situations, the amount of free ioncharges in the solution can be altered to change the degree of variationin alignment between layers.

Auxiliary Molecules

The methods described herein can include the use of one or moreauxiliary molecules, e.g., collagen modulating molecules such asextracellular matrix molecules. Such molecules include, but are notlimited to, proteoglycans (such as perlecan, versican, syndecan,decorin, lumican, and biglycan), proteoglycan core proteins,glycosaminoglycans (such as hyaluronic acid, chondroitin-4 sulfate,chondroitin-6 sulfate, dermatan sulfate, heparin, heparin sulfate, andkeratan sulfate), Type V collagen, fibronectin, or any molecule thatcompetes with collagen for available water (such as polyethyleneglycol). Such molecules can be added to a solution containing collagenmonomers prior to or following the precipitation of collagen asdescribed herein.

Methods of Strain Stabilization, Monomer incorporation and EnzymaticDegradation of Collagen

Some of the methods described herein are based, at least in part, on thediscovery of a strain-dependent mechanism that can modulate collagenfibril susceptibility to enzymatic degradation. This mechanism canproduce a physicochemical change at the matrix level that is bound tofibril strain. Based on this “strain-stabilization of collagen”mechanism, tensile strains can provide a robust signal, leading to aload-controlled differential degradation (catabolism) of collagen inextracellular matrix. Further, tensile strains on collagen fibrils canprovide a signal, leading to the incorporation of collagen monomers intoloaded fibrils (this is monomer incorporation). In some instances, theadaptive remodelling response of load-bearing ECM can be controlled bycollagen and its complement enzymes (e.g., bacterial collagenase, MMPs,and cathepsins), which couple the control signal (i.e., mechanical load)to a physicochemical change in the collagen molecules or fibrils. Inaddition, this mechanism can relieve fibroblasts of the burden of“knowing” which fibrils to degrade during remodelling and which toreinforce. Based on this mechanism, load-stimulated fibroblasts canproduce a load-adapted morphological change during, e.g., epigeneticconnective tissue remodelling, repair, homeostasis and disease.

In some instances, collagen is precipitated as described herein, and thecollagen organization is further refined by subjecting the initialcollagenous construct to cross-linking, mechanical strain, and/orenzymes to cull unwanted (unstrained) fibrils (see, e.g., Ruberti etal., Biochem. Biophys. Res. Commun. 336:483-489 (2005)).

Mechanical strain can be applied to collagen fibrils using, e.g., amicrochamber (see, e.g., FIG. 25). In such methods, the collagen can befixed to grips in a microchamber by, e.g., direct clamping or byadhesives (such as cyanoacrylates). In some situations, the collagen isaffixed to functionalized micropipettes as described herein. During theloading of mechanical strain, auxiliary molecules can optionally beincluded. In addition, hydroxyapetite and noncollagenous proteins can beadded to calcify the system during loading.

In some situations, prior to the loading of the construct, collagenfibrils are cross-linked to facilitate strain production. Any suitablecrosslinking agent known in the art can be used including, withoutlimitation, formaldehyde, hexamethylene diisocyanate, glutaraldehyde,polyepoxy compounds, gamma irradiation, and ultraviolet irradiation withriboflavin. The crosslinking can be performed by any known method (see,e.g., Bailey et al., Radiat. Res. 22:606-621 (1964); Housley et al.,Biochem. Biophys. Res. Commun. 67:824-830 (1975); Siegel, Proc. Natl.Acad. Sci. U.S.A. 71:4826-4830 (1974); Mechanic et al., Biochem.Biophys. Res. Commun. 45:644-653 (1971); Mechanic et al., Biochem.Biophys. Res. Commun. 41:1597-1604 (1970); and Shoshan et al., Biochim.Biophys. Acta 154:261-263 (1968)).

Enzymes

The methods described herein can be used to “sculpt” collagenous ECMsthrough application of mechanical load and also exposure tocollagen-degrading enzymes including, without limitation, collagenase(e.g, bacterial collagenase), cathepsin, and matrix metalloproteases(MMPs). Thus, in some instances, collagen is contacted with acollagen-degrading enzyme and simultaneously subjected to mechanicalload.

One non-limiting useful collagen degrading enzyme is bacterialcollagenase (BC). BC attacks the triple helical domain of fibrillarcollagen non-specifically at multiple sites. Another non-limiting usefulcollagen degrading enzyme is a matrix metalloprotease (MMP). In mammals,fibroblasts degrade matrix by expressing MMPs, which cleave collagen ata specific location three-quarters of the distance from theamino-terminus MMP degradation of collagen proceeds by a ratchetmechanism, whereby the enzyme moves along collagen fibrils in steps,alternately binding to and cleaving the available monomers. MMPs cleavethe collagen triple helix en bloc at a site that is at the interface ofa loose, less rigid portion of the collagen triple helix (due to a lowerhydroxyproline content) and a tighter portion of the triple helix. Forcleavage to occur, the MMP catalytic domain also “unwinds” the collagentriple helix locally to gain access to a single alpha chain, while thehemopexin-like domain orients and destabilizes the collagen. While notwishing to be bound by theory, tensile mechanical loads may affect, e.g,reduce, the rate of MMP cleavage by one of the three followingmechanisms: 1) reduction of configurational entropy of the binding site,which increases the energy to unwind and orient the collagen, 2) bindingsite distortion, which reduces the binding affinity, and 3) causing arelative shift in alpha chain registration, which makes cleavingdifficult.

The cleavage rate of fibrillar collagen by MMPs or the rate of monomerincorporation into fibrils can be affected, e.g., reduced, by tensilemechanical loads. The load state of the matrix dictates which collagenfibrils are affected by the collagen degrading enzymes. As a corollary,newly secreted collagen intermediate filaments or monomers arepreferentially incorporated into loaded fibrils and gain resistance toMMP degradation. Thus, these methods can be used to preserve onlyfibrils under strain, resulting in a matrix that is adapted to theapplied load.

Methods of Engineering Tissues

Collagen produced by the methods described herein can be used toengineer tissues or organs including, but not limited to, bone, dentalstructures, joints, cartilage, skeletal muscle, smooth muscle, cardiacmuscle, tendons, menisci, ligaments, blood vessels, stents, heartvalves, corneas, ear drums, nerve guides, tissue or organ patches orsealants, a filler for missing tissues, sheets for cosmetic repairs,skin (sheets with cells added to make a skin equivalent), soft tissuestructures of the throat (such as trachea, epiglottis, and vocal cords),other cartilaginous structures (such as articular cartilage, nasalcartilage, tarsal plates, tracheal rings, thyroid cartilage, andarytenoid cartilage), connective tissue, vascular grafts and componentsthereof, and sheets for topical applications or for repair orreplacement of organs (such as livers, kidneys, and pancreas).

In some situations, the collagen is produced having a predeterminedshape, such as a predetermined shape dictated by external and internaltemplates described herein. In specific instances, the templates can beshaped, for example, in the shape of a nerve guide, skin or musclepatch, fascial sheath, vertebral disc, knee meniscus, ligament, tendon,or a vascular graft for subsequent use in vivo. The collagen can also beshaped to fit a defect or site to be filled, e.g., a site where a tumorhas been removed or an injury site in the skin (e.g., a cut, a biopsysite, a hole or other defect) or to reconstruct or replace a missing orshattered piece of bone. The methods described herein allow for greatflexibility and the ability to customize the collagen to virtually anyshape needed. Specific geometries include, but are not limited to, acylindrical shape, a flattened oval shape, capillary tubes (as intendon), concentric cylinders (as in artery, annulus fibrosus, lamellarbone) and nested hemi-spheres (as in cornea).

In some instances, further shaping can be achieved by manuallyprocessing the formed collagen. For example, formed collagen can besutured, sealed, stapled, or otherwise attached to one another to form adesired shape

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

EXAMPLES Example 1 In Vitro Production of Organized Collagen LamellaeUsing a Scaffold-Free Model of Corneal Stromal Development

An in vitro model of corneal stromal development was developed asfollows. Briefly, primary human corneal fibroblasts (by migration fromcorneal explants in culture) were grown to confluence on a transwellmembrane and stimulated to produce matrix with a stable form of ascorbicacid (Guo et al., Invest. Ophthalmol. Vis. Sci. (2007),48(9):4050-4060). This scaffold-free approach allowed the fibroblasts toself-organize and then stratify, and is the first culture system capableof producing significant quantities of organized lamellae that aremorphologically similar to in vivo lamellae in that they alternate indirection. It also shares some features of developing systems, includingcell organization and a relatively high ratio of cell volume to matrixvolume (at least initially).

The data obtained demonstrate that highly-organized collagen wassynthesized between two confluent monolayers of fibroblast cells. Themonolayers appeared to separate from one another, as organized matrixwas secreted between them. FIGS. 1A-1B compare in vivo development ofcollagen organization in the primary human fibroblast culture to that ofa developing primate. Both images demonstrated flattened fibroblasts(dedifferentiated human fibroblasts for the in vitro study andmesenchymal fibroblasts for the in vivo development study) and collagenfibrils that alternated in direction in subsequently deposited lamellae.The thickness of the lamellar collagen arrays was about 1μ-2μ in bothsystems (which may be an intrinsic length scale for collagen rotation).

The fibroblasts produced 25μ of aligned collagen lamellae (each about 2μm) within a thicker construct (36 μm) by four weeks as measured bydifferential interference contrast microscopy (DIC). The model wasextended to 12 weeks and reached steady state by 8 weeks (50 μm thickwith 25 μm of organized matrix), but appeared to begin to lose collagenfibrils by 12 weeks.

The organized collagenous lamellae were confined between the boundingcell layers, which may show that confinement is important. Further, thelamellae were approximately 2 μm thick before changing direction, whichmay show the existence of an intrinsic organizational length scale.

Example 2 De Novo Production of Sheets of Organized Collagen byConcentration and Confinement

To generate local, long-range and three dimensional fibril organization,a method was developed using the organizational information encoded intothe collagen triple helix. Using this method, confined, patterned arraysof monomers condensed into organized arrays of fibrils upon enzymaticcleavage of collagen propeptides.

Methods

Preparation of Collagenous Constructs—Method I.

3 mg/ml solution of collagen (Inamed biomaterials, Fremont, Calif.) wasdialyzed against 40% solution of polyethylene glycol (PEG) (20 kMWCO,Sigma) at 4° C. to reach the concentrations in the range of 140mg/ml-200 mg/ml (medium concentration). The collagen solution wasneutralized (pH 4.5-7) with 1.5 M Trizma based solution (Sigma-Aldrich,St. Louis, Mo.), confined between two coverslips, transferred into a 37°C. incubator, and stored for 6 hrs prior to assessment.

Preparation of Collagenous Constructs—Method II.

A medium concentration solution of collagen (prepared as describedabove) was transferred into a 3.5 kMWCO dialysis cassette (ThermoScientific, Rockford, Ill.) and left in 40% solution of PEG overnight at4° C. to reach concentrations in the range of 200 mg/ml-400 mg/ml. Toneutralize the collagen solution, the cassettes were transferred into aneutralized PEG solution (using trizma base) and stored up to 2 wksprior to assessment.

Primary Human Corneal Stromal Cell-Derived Constructs.

Cell-derived constructs were produced as described in Guo et al.(Invest. Ophthalmol. Vis. Sci. 48:4050-4060 (2007)) on disorganizedcollagenous mats (Ren, et al. Dev. Dyn. 237:2705-2715 (2008)).

Transmission Electron Microscopy (TEM).

Constructs were fixed overnight in Karnovsky fixative (2.5%glutaraldehyde, 2.5% formaldehyde, 0.1 M cacodylate buffer), washed with0.1 M buffer, post-fixed in 2% osmium tetroxide, and dehydrated indegraded ethanol. The samples were infiltrated and embedded in modifiedspurr resin (as described in Ellis, Microscopy Today 14:32 (2006)). 60nm cross sectional and en face sections were cut using a diamond knifeultramicrotome (Ultra Cut E Microtome; Reichert, Depew, N.Y.) andstained with 5% uranyl acetate and Reynolds lead citrate. The sectionswere viewed and photographed with a transmission electron microscope(JEM 1010; JEOL, Tokyo, Japan).

Scanning Electron Microscopy (SEM).

SEM analysis was conducted as described in Example 3.

Differential Interference Contrast (DIC) Microscopy.

The long range organization of the collagen fibrils was investigatedusing DIC (Guo et al., Invest. Ophthalmol. Vis. Sci. 48:4050-4060(2007)). The collagenous constructs were transferred between twocoverslips and placed on the stage of an inverted microscope (TE2000U;Nikon). The alignment of collagen fibrils was studied using series ofin-plane and Z-stacks.

Results

As shown in FIG. 2, primary human corneal fibroblasts naturally formedconfluent and generally orthogonal sheets on two-dimensional surfaces.The following experiment was conducted to determine whether confinementand concentration of monomers leads to the production of organizedarrays of collagen fibrils, the following. Triple helical domains ofType I collagen (atelo-collagen monomers extracted from bovine dermis(PURECOL®)) were concentrated to liquid crystalline levels at thefollowing two different concentrations. Low concentration (LC) was 140mg/ml-200 mg/ml and high concentration (HC) was 280 mg/ml-400 mg/ml. Thecollagen monomers in the resulting viscous solutions were then“precipitated” to form fibrillar structures while confined between twosurfaces.

FIG. 2D shows the cross-section of the lamellar-like stratification of atypical LC de novo construct produced by confinement and concentrationof monomer followed by fibillogenesis.

Differential interference contrast (DIC) z-scan optical imaging was inagreement with the thick section data and revealed that the de novoconstructs possessed multiple “lamellae” within which the fibrillarmatrix was uniformly aligned over long distances in the x-y plane on theorder of hundreds of microns (FIG. 3A). For the LC experiments, thespontaneously formed lamellae were 16.6μ±6.3μ, thick and theirconstituent fibrils changed direction en mass at an average angle of50±24 degrees.

DIC images of the LC constructs were qualitatively compared withconstructs produced by fibroblasts in vitro. In general, the lamellarorganization in the LC de novo constructs compared favorably with thematrix alignment found in PHCSC-derived stromal constructs (FIG. 3). InPHCSC-derived constructs, synthesized lamellae are generally 2μ-5μ thickand appear to change direction by 90°. This was observed when directionchanges were clearly observable by DIC (see FIGS. 3D and 3F). In thenormal human cornea, collagen lamellae are approximately 2μ thick, andthe angle between successive lamellae is also generally 90° (asdescribed in Meek et al., Exp. Eye Res. 78:503-512 (2004)). Thus, the LCde novo constructs synthesized comprised thicker aligned lamellarstructures that changed direction more arbitrarily than native tissue.

Standard transmission (sTEM) and scanning (SEM) electron microscopy ofthe de novo constructs in cross-section corroborated the results of theDIC imaging by revealing alternating arrays of aligned fibrils in ersatz“lamellae”. Low magnification sTEMs of the constructs produced from LC(FIG. 4B) and HC (FIG. 4C) collagen bore a striking resemblance to sTEMsof the normal human cornea (FIG. 4A) (see, Komai et al., Invest.Ophthalmol. Vis. Sci. 24:543-556 (1983)). Cross-sectional SEM alsoconfirmed the high fibril density, high-degree of fibril alignment andlamellar structure in constructs produced from HC collagen (FIG. 6A).The sTEMs also showed that the spontaneously formed lamellae varied inthickness (FIG. 4D).

Taken together, the low magnification sTEM, SEM and DIC confirm thatdense, aligned fibrillar structures spontaneously precipitated fromconfined, concentrated monomers. Confined collagen appears to becooperative in that lamellae appeared to be spontaneously formedparallel to the confining surfaces.

Fibroblasts may play a role in providing additional “guidance cues” todirect the forming collagen lamellae. Fibroblasts in the boundingconfluent layers may provide external guidance cues (e.g, elongated cellshape) while embedded fibroblasts may control local directionality ofthe monomers via internal guidance cues (e.g, elongated cell shape andfilipodial extensions). In PHCSC derived constructs, collagen fibrilswere often seen in co-alignment with the long axis of the fibroblastbody. FIG. 5A shows two adjacent lamellae produced by PHCSCs wheredirection of the fibrils and of the embedded cells (which areco-aligned) changes abruptly. FIG. 5B demonstrates that the direction ofcollagen fibrils can be locally modified by the presence of ahigh-aspect-ratio object (such as a glass microcylinder). Thus, thecombination of geometric confinement and internal guidance cues (spacedappropriately) may be used to fully control the orientation of collagenfibrils precipitated from dense solutions of collagen monomers.

The cross-sectional morphology of precipitated fibrils was also examinedFIGS. 6A and 6B are low magnification and high magnification sTEMs of anarray of collagen fibrils in cross-section that were precipitated fromHC collagen monomer solutions. The higher magnification image of the denovo construct fibrils revealed generally small diameter, highlypolydisperse and irregular “fused” fibrils. Auxiliary ECM molecules suchas proteoglycans, glycosaminoglycans or other collagens may aid in theformation of uniform diameter or circular fibrils.

In the plane of the de novo constructs, TEM generally confirmed the DICimages. Large areas of complete fibril alignment parallel to theconfining surfaces were seen (FIGS. 7A and 7B). In LC constructs,fibrils sometimes adopted a “wavy pattern”, suggesting the production ofopen space as monomers were incorporated into the aggregating fibrils(FIG. 7A). In HC constructs, the collagen was generally more tightlypacked and highly-aligned parallel to the confining surface (FIG. 7B).At high magnification, collagen fibrils in longitudinal section werethin (about 20μ), tightly packed and highly aligned (FIG. 7C). Directcomparison to PHCSC-synthesized collagen (FIG. 7D) demonstrated that thefibrils in the de novo construct were smaller and did not have clearD-periodic banding (though some striations were seen).

These results demonstrate that adequate information to produce bothshort and long-range fibrillar order is provided by the confininggeometry and the triple-helical domain of the collagen molecule. Theseresults further demonstrate that the triple helix in atelo-collagencarries long-range structural information that can be leveraged simplyby confinement and concentration. This can relieve fibroblasts of theneed to “stitch together” complex, highly organized 2-D+ structures likethe cornea. Rather, fibroblasts confine and concentrate collagen monomerwhile providing low-energy directional cues (such as active cellattachments to the matrix or merely the high aspect ratio “spindle”shape fibroblasts typically employ). Thus, cell organization may occurfirst, followed by organized matrix production, as was seen in thePHCSC-derived constructs.

Example 3 Geometric Guidance Cues Influence the Organizational Behaviorof Collagen Fibrils Precipitated from Liquid Crystalline (LC) PhaseSolutions of Monomer

The concentration and precipitation of collagen monomers in theappropriate guiding geometry leads to the formation of fibrillarstructures with organizations similar to those found in naturalload-bearing tissues.

A. The Relationship Between Concentration and the OrganizationalBehavior of Collagen Molecules

Both long and short range organization of collagen molecules are afunction of monomer concentration and confining surface separation;monomer organization is readily translated to precipitated fibrillarorganization.

Methods

Cold, acidic solutions of the atelo-collagen Type I monomers (Inamed,Fremont, Calif.) are dialyzed against 40% solution of 20 kMWCOpolyethylene glycol (PEG) using 3.5 kMWCO dialysis tubing (SpectrumLabs, Rancho Dominguez, Calif.) to obtain the concentrations listed inTable 1.

TABLE 1 Parameters for Channel Depth and Concentration Depth (μm) Conc(mg/ml) 0.5 2 4 10 100 500 50 ✓ ✓ ✓ ✓ ✓ ✓ 100 ✓ ✓ ✓ ✓ ✓ ✓ 150 ✓ ✓ ✓ ✓ ✓✓ 200 ✓ ✓ ✓ ✓ ✓ ✓ 250 ✓ ✓ ✓ ✓ ✓ ✓

The solution is dispensed into guiding geometries (channels of varyingdepth etched into a coverslip as shown in FIG. 8). Briefly, thetemplates and microchannels are manufactured by spin coating a thinlayer (120 nm) of PMMA onto the substrates (e.g, coverslips or silicon).In the case of a coverslip, an additional 5 nm layer of gold coating isadded using an E-beam evaporator. The pattern of templates is written onthe substrates using a supra 25 Zeiss SEM device equipped with a J, C,Nabity ver. 9—lithography and pattern generation system. The patternsare developed in a solution of MIBK and are etched to the desired depthusing an ICP ether Dry plasma etch reactor.

Loaded channels are covered with a blank coverslip and stored at 4° C.for up to 96 hrs. Channel depths are chosen to correspond to naturallength scales of the system—collagen molecule length, single andmultiple tissue lamellar thickness (about 2 μm to about 100 μm), totalthickness of a representative tissue (e.g., corneal stroma).

The triple helical domain can dominate organizational behavior ofcollagen at high concentration. However, the short non-helical terminaltelopeptides (which may be important for cross-linking/mechanicalstrength) may influence the liquid crystalline organization. Thus, asubset of the experiments that produce the best organization arerepeated using tropocollagen.

Acid-extraction of collagen preserves intact telo-peptides. Briefly,tropocollagen is obtained from bovine corneas following the methoddescribed by Trinkaus-Randall et al. (Invest. Ophthalmol. Vis. Sci.32:603-609 (1991)).

Collagen fibrillogenesis is facilitated by neutralization and warming ofthe dense solution. Neutralization is accomplished by transfer ofconfined collagen in situ coverslips into a neutralized mixture of PEGin 1.5 M Trizma base solution (Sigma, St. Louis, Mo.) and allowing freeion exchange between two solutions through the porous photoresist. Thecollagen phase pH is monitored using pH sensitive dyes (Sigma, St.Louis, Mo.). Following neutralization, the monomers are incubated insitu at 37° C. for up to 30 days. Precipitated fibril alignment strengthand lamellar angle changes are assessed by Differential InterferenceContrast (DIC) microscopy and polarizing light microscopy (PLM) at 1day, 2 days, 3 days, 7 days, 14 days, and 30 days.

DIC Microscopy is carried out as described by Petroll and Ma (CellMotility and the Cytoskeleton 55:254-264 (2003)), with the exceptionthat a Nikon TE-2000E inverted microscope equipped with a 60×1.45 NA oilimmersion objective (Nikon, Melville, N.Y.) and Coolsnap EZ camera(Photometrics, Tucson, Ariz.) are used.

The large-scale organization and LC phasing of the collagen molecules isinvestigated using polarizing light microscopy (PLM) at time points (1hr, 3 hrs, 6 hrs, 12 hrs, 24 hrs, 48 hrs, and 96 hrs). QuantitativeOptical PLM is performed using an upright Nikon microscope equipped witha center-locked rotating stage and a Cri Abrio Micro Imaging system.

The ultrastructural organization and morphology of the fibrils (e.g.,D-banding, fibril diameter, and fibril-to-fibril spacing) areinvestigated by standard TEM (sTEM) and Quick Freeze Deep Etch (QFDE).All collagenous constructs are processed routinely for sTEM as describedin Guo et al., Invest. Ophthalmol. Vis. Sci. 48:4050-4060 (2007), withthe following exceptions. 1% osmium tetroxide is used instead of 2%, andimages are viewed on a TEM (JEOL JEM-1000, Tokyo, Japan). Confinedsamples are fixed by transferring them in situ to a fixative reservoirat 4° C. ON to allow for complete diffusion of fixative molecules. ForQFDE imaging, samples are prepared as described in Guo et al. (supra);however, a custom modified CFE-40 Freeze fracture/freeze etch unit(Cressington Scientific, Watford, UK) is used, and images are taken on aJEOL JEM-1000 (Tokyo, Japan).

The tensile modulus of the construct is evaluated. All mechanicalanalyses are performed on collagenous constructs that are crosslinkedusing the primary fixation method described. Tensile material propertiesof the collagenous matrix are measured as described in Wan et al., ThinSolid Films, 425:150-162 (2003), with additional constraints oncalculation of a buoyancy correction for sample immersion in 1×PBS.In-plane Young's modulus is reported.

Results

A clear, repeatable relationship is expected between concentration,surface separation and the organization (length scale and average angleof spontaneous direction changes) of collagen monomers in densesolutions. Monomer organization is directly translated to precipitatedfibrillar organization, and the ability to predict and control matrixorganization are established.

B. External Guidance Cues Influence the Organization of PrecipitatingCollagen Fibrils

A close relationship exists between the orientation of elongatedfibroblast cells in a confluent sheet and the organization ofsynthesized fibrillar arrays. External guidance cues (e.g., patterns onconfining surface) with geometric scales similar to an elongated cellcan influence the organization of adjacent collagen fibrils precipitatedfrom dense solutions. This effect can propagate surface organizationdeep into the bulk collagen solution. A measure of the influence of ageometric feature on the bulk LC material is the “persistence length”,defined as the maximum distance from the feature where fibril alignmentis affected. To calculate persistence length, a z-stack (0.5 μm step) ofimages is captured. At each position, the angle of alignment and thestrength of alignment is calculated to produce a 3-D field adjacent tothe guidance cue. The “persistence length” is defined as the point wherethe angle of alignment is within 15% of the direction of the guidancecue, and the calculated strength of alignment greater than 95%.

Methods

Various high-aspect-ratio prismatic rectangles are formed on the surfaceof a coverslip (as described in Part A above). Table 2 shows theparameters for the length (L), width (w), and height (h) of the externaltemplates. The scale of these dimensions match the natural length scalesin ECM from monomer length to elongated cells. Number of runs pergeometrical feature=3.

TABLE 2 Parameters of External Templates Width (w) and Length (L, μm)Height (h, μm) 0.3 2 5 10 0.3 ✓ ✓ ✓ ✓ 1 ✓ ✓ ✓ ✓ 2 ✓ ✓ ✓ ✓ 4 ✓ ✓ ✓ ✓ 8 ✓✓ ✓ ✓ 15 ✓ ✓ ✓ ✓

The rectangles serve as a single templating feature (FIGS. 9A-9C). Theoptimal concentration of atelo-collagen in solution (determined in PartA above) is then introduced onto the coverslip and confined from abovewith a featureless glass plate.

The feature that produces the maximum persistence length is multiplyreproduced on the surface of a single coverslip with a spacing of twicethe maximum persistence length found in the single feature experiment(FIG. 9B). Dense atelo-collagen solution is introduced and confined fromabove with a featureless glass plate. The persistence of thetemplate-induced 3-dimensional organization into the bulk collagen isassessed. The alignment of collagen fibrils is quantified from DICimages using the edge detection method previously described by Chaudhuriet al. (Pattern Recogn. Lett. 14:147-153 (1993)). Briefly, the DIC imageis convolved with two directional masks, h_(x) and h_(y), which resultsin the matrices G_(x) and G_(y). The gradient vector is calculatedaccording to: G=G_(x) ²+G_(y) ² and θ=π/2+tan⁻¹(G_(y)/G_(x)).

The previous experiment is repeated with a second identical templatecoverslip (rather than a featureless surface) oriented perpendicular tothe first to enclose the chamber (see FIG. 9C). The separation distancebetween the coverslips is reduced until the persistence length from eachsurface template overlaps. This experiment is repeated usingtropocollagen.

Long range organization and alignment of precipitated fibrils isinvestigated using DIC and PLM as described in Part A above. The shortrange organization and morphology of the fibrils (e.g., D-banding,fibrils' diameter, and fibril-to-fibril spacing) are investigated bysTEM and QFDE imaging as described in Part A above. The tensile modulusof each construct is evaluated as described in Part A above. Eachexperiment is repeated 3 times.

Results

Specific external template parameters (geometries) are established thatmaximize the propagation of fibrillar organization into the bulk LCcollagen.

C. Internal Template Guidance Cues Influence the Organization ofPrecipitating Collagen Fibrils

Fibroblasts may control the alignment of collagen fibrils with theircell bodies or by extending filopodia. Whether internal templates ofdimensions similar to filopodia (about 300 nm) can influence theorganization of collagen over both short and long length scales isdetermined

Methods

The internal template comprises a prismatic square formed on a thinsilicon wafer (FIG. 10A) and is placed between two thick gaskets. Highconcentration atelo-collagen is dispensed in the area surrounding thetemplate and confined from both sides with two featureless coverslips.The fibrils are precipitated using the method described in Part A above.The width and thickness of internal templates are chosen based on thediameter of filopodia (300 nm and 500 nm) and elongated fibroblasts (1μm and 5 μm). The length of all prismatic squares are 0.5 cm.

A series of parallel internal templates of similar geometries and aspacing of twice the most effective persistence length found using asingle internal template are produced in a thin silicon wafer. Similarto the method used for a single internal template, atelo-collagen isdispensed, confined, and precipitated between two coverslips.

The previous method is repeated using two internal templating surfaceswith similar geometries and perpendicular alignment. The spacing betweenthe features on a single template is twice the persistence length foundin the previous section. The distance between each of the internaltemplates to the adjacent confining coverslip is the same as thepersistence length. This arrangement appears to result in completeorganization of the fibrils between the coverslips.

DIC and PLM are used to investigate fibrillar organization across theconstruct, as described in Part A above. Alignment is evaluated usingsTEM, and QFDE is used to further investigate the morphology andalignment of the resulting fibrils (as described in Part A above). Thepersistence length and the tensile modulus of each construct areevaluated as described in Part A above. Experiments are repeated 3times.

Results

A combination of template geometry/spacing and collagen concentrationare found that predictably control fibrillar organization at largedistances from the template.

The natural length scale(s) associated with collagen organization atliquid crystalline concentrations (including characterizing spontaneousdirection changes) are elucidated, along with a measure of the extent ofthe ability to control fibril organization via internal and externalgeometric guidance cues. This method provides a full assessment of thefeasibility of utilizing the liquid crystalline behavior of collagen,along with guidance cues, to produce native-like fibrillar organization.Since surface features have the capacity to propagate organization overlarge distances into LCs, long-range control over the collagen isachieved.

Example 4 The Effect of Biologically Relevant Geometries on theOrganization of Precipitated Fibrils

The organization of precipitated fibrils from LC collagen monomers invarious native developing tissues is in part determined by theirnaturally confining geometries. Such long-range influence is possiblebecause of the natural tendency of the constituents of LCs to behave asa single unit.

A. Confined Collagen Monomers within a Cylinder

Methods

Monomeric solutions of atelo-collagen are concentrated and injected intoglass capillary tubing (BD, St. Louis, Mo.) of dimensions defined inTable 3.

TABLE 3 Parameters for Inside Diameter (R) and Length (L) of CapillaryTubing Length (L, cm) ID (R, mm) 0.5 1 5 0.5 ✓ ✓ ✓ 1 ✓ ✓ ✓ 2 ✓ ✓ ✓

Following injection, the ends of the tubing are blocked using 3.5 KMWCOdialysis tubing (Spectrum Labs, Rancho Dominguez, Calif.).Fibrillogenesis proceeds by neutralization through the dialysis tubingand warming as described in Example 3. A limited set of runs usingtropocollagen are performed in the geometry that generates the bestalignment.

After fibrils are precipitated (as described in Example 3), DICmicroscopy is used to investigate the long-range organization ofcollagen fibrils. TEM and QFDE are used to evaluate morphology and shortrange organization.

Results

Confining a high-concentration solution of collagen molecules into athin cylindrical tube (e.g., capillary tubing) causes the collagenmolecules and the resulting precipitated fibrils to align in the samedirection as the long axis of the cylinder (analogous totendon/ligament). The resulting assembly of collagen molecules into longand parallel fibrils is similar to tendon fascicles.

B. Confined Collagen Monomers within Concentric Cylinders

Methods

Highly-concentrated atelo-collagen molecules are injected into the spacebetween concentric glass cylinders (BD, St. Louis, Mo.) (FIG. 11) withradius difference (r) (Table 4).

TABLE 4 Parameters for the Thickness (r) Between Two Cylinders andLength (L) Length (L, cm) Thickness (r, mm) 0.5 1 5 0.5 ✓ ✓ ✓ 1 ✓ ✓ ✓ 2✓ ✓ ✓ 5 ✓ ✓ ✓

The ends of the tubes are blocked using dialysis membrane, and thecollagen solution is neutralized and warmed as described in Example 3.The geometrical parameters that provide the best result are tested withtropocollagen.

DIC is used to evaluate the organization and alignment of the collagenfibrils in large scale. TEM and QFDE are used to investigate themorphology and short range organization of the fibrils. Experiments arerepeated three times.

Results

Confining collagen monomers in a thin space between two concentriccylinders causes precipitating fibrils to assemble into parallellamellae tangent to the cylinder surface. The precipitating collagenfibrils are seen to organize into concentric lamellae similar to thecollagen organization in annulus fibrosus.

C. Cornea-Like Collagen Organization

Methods

Atelo-collagen monomers are concentrated and confined between twoconcentric hemispheres (FIG. 12) that are obtained from McMaster-Carr(Princeton, N.J.). The dimensions are chosen to provide biologicallyrelevant thicknesses (r) and radii of curvature, L (Table 5).

TABLE 5 Parameters for Hemisphere Radius and Thickness Between TwoHemispheres Radius (L, cm) Thickness (r, mm) 0.5 1 1.5 0.5 ✓ ✓ ✓ 1 ✓ ✓ ✓2 ✓ ✓ ✓ 5 ✓ ✓ ✓ 10 ✓ ✓ ✓

One hemisphere is fixed onto a coverslip and filled partially withcollagen solution. The second hemisphere is positioned to produce theappropriate gap. The open gap is sealed with dialysis tubing and fibrilsare precipitated by neutralization and warming similar to the methoddescribed in Example 3. The geometry that produces the best organizationis used to repeat this experiment with tropocollagen.

DIC microscopy is used to investigate the long-range organization of thefibrils and lamellae. sTEM and QFDE are used to investigate themorphology and short range organization of the fibrils. Experiments arerepeated 3 times.

Results

Confining a high concentration of collagen molecules into limited spacewith corneal-like curvature results in the formation of highly alignedfibrils arranged into orthogonal layers similar to native corneallamellae.

The large scale confining geometry plays a significant role in theorganization of precipitated collagen fibrils. The large length scalesover which liquid crystals can be manipulated are sufficient to producestructures in precipitated collagen that mimic in vivo fibrillarorganization.

Example 5 Auxiliary ECM Molecules Influence Collagen Fibril Morphologyand Spacing

The collagen triple helix contains adequate information to produce bothshort and long-range organization. Collagen fibril morphology (such asdiameter and D periodicity) and fibrillar spacing may be under thecontrol of auxiliary ECM proteins (such as proteoglycans andglycosaminoglycans). The influence of controlling ECM moleculesglycosaminoglycans, hyaluronic acid, proteoglycan core proteins, intactproteoglycans and collagen Type V on the morphology and structuralorganization of Type I collagen fibrils precipitated from densesolutions are determined

The presence of highly-sulfated GAG chains or HA increases the rate offibrillogenesis due to the co-nonsolvency effect that is produced by thepresence of a high fixed negative charge density. The effects of threemajor non-collagenous components found ubiquitously throughout variousconnective tissues (keratan and chondroitin sulfates (KS and CS) and HA)are assessed. To demonstrate that the effect is physicochemical (ratherthan specific), a substitute hygroscopic molecule is also tested.

A. Highly Sulfated GAGs Fixed Charge Density Influences the Rate of TypeI Collagen Fibrillogenesis

Methods

Purified bovine corneal KS and shark cartilage CS are obtained fromSigma (#K3001 and #C4384 respectively). Each GAG is serially diluted andpipetted into optimized cold, cholesteric collagen mixtures prior toconcentration, and the resulting mixture is processed as described inExample 3 to allow assembly. The GAG content of concentrated collagensolutions is normalized to physiological range as outlined in Table 6.

TABLE 6 KS/CS Single GAG Addition Conditions Post Concentration Hrs ofassembly GAG addition 6 12 24 36 48 72 120 μg/ml ✓ ✓ ✓ ✓ ✓ ✓ 225 μg/ml ✓✓ ✓ ✓ ✓ ✓ 500 μg/ml ✓ ✓ ✓ ✓ ✓ ✓ 750 μg/ml ✓ ✓ ✓ ✓ ✓ ✓ 1000 μg/ml ✓ ✓ ✓ ✓✓ ✓ 7000 μg/ml ✓ ✓ ✓ ✓ ✓ ✓ 20 mg/ml ✓ ✓ ✓ ✓ ✓ ✓

The rate of collagen fibril assembly is assessed using DIC microscopy(n=3) at strategic timepoints based on the developmental sequence of thechick cornea. Cuprolinic blue staining in conjunction with sTEM is usedto assess fibril banding and GAG interaction (n=1). Briefly,high-resolution visualization of the sulfated GAGs is accomplished by enbloc cuprolinic blue labeling as described in Scott (Coll. Relat. Res.5:541-575 (1985)). Stained sections are imaged on JEOL JEM-1000 (Tokyo,Japan).

GAG chain assimilation is also tested in the entire construct on thelight level scale by immunofluorescence microscopy against high and lowKS (5D4 and 1B4) and CS (4C3 and 7D4) sulfation motifs (n=1).

To determine the assimilation of controlling molecules, constructs areoriented horizontally and vertically in embedding medium (for in planeand transverse sections), frozen, and sectioned on a cryostat. Labellingof the sections with antibodies raised against HA, highly-sulfated andlesser sulfated GAG motifs, and all PG core proteins are carried out asdescribed in Young et al. (Invest. Ophthalmol. Vis. Sci. 48:3083-3088(2007)). Constructs are observed using a Nikon Eclipse TE2000-E (Nikon,Japan) microscope at 25× magnification to achieve full thickness images.Antibodies and antibody specificities for immunofluorescence andimmuno-electron microscopy are listed in Table 7.

TABLE 7 Antibody Description Epitope Pre-treatment 5D4 Mouse monoclonalLinear penta-sulfated sequences of N-acetyl None - reacts with nativeIgG lactosamine disaccharides of KSPGs with both epitope GalNAc and Galsulphated (Caterson et al. 1985, Mehmet et al., 1986) 1B4 Mousemonoclonal Linear tetra-sulfated sequences of N-acetyl None - reactswith native IgG lactosamine disaccharides of KSPGs (Mehmet et epitopeal.., 1986) 4C3 Mouse monoclonal Chondroitin-6-sulfate with non-reducingtermination None - reacts with native IgG of GlcAβ1,3GalNAc6S- (Plaas etal., 1997) epitope. Can be used in conjunction with C'ase ABC 7D4 Mousemonoclonal Lesser sulfated combinations of sulfated and non- None -reacts with native IgG sulfated disaccharide isomers in native CSepitope (Caterson et al., 1995). Proposed as a marker for OA (Carlson etal., 1995) Lum⁻¹ Mouse monoclonal Lumican core protein, amino acidsequence K'ase I/II/endo-β-gal to IgM unknown remove KS side chains 70.6Mouse monoclonal Amino acid residues 50-65, termed the N-terminal None -reacts with native IgG cysteine cluster region of decorin (Sawada etal., epitope. C'ase ABC aids 2002) immuno-EM. Anti-HA Mouse monoclonalHyaluronic acid, exact epitope unspecified None - reacts with native IgG(Immunology Consultants Laboratory Inc.) epitope Anti-PEG Rabbitpolyclonal Terminal methoxy group of the PEG molecule None - reacts withnative IgG (Epitomics) epitope

Results

The addition of sulfated GAG chain fixed negative charge densities intoa concentrated collagen solution expedites fibril formation incomparison to untreated controls. The greater the water attractive andabsorptive tendencies, the more pronounced the co-nonsolvency effect. Invivo, corneal GAG chains undergo increasing net sulfation duringembryonic development, and the water retentive and attractive propertiesof these KS and CS/DS GAGs are not equal. Control of the co-nonsolvencyeffect allows control of the rate of fibrillogenesis de novo.

B. Unsulfated HA Influences the Rate of Type I Collagen Fibrillogenesisand Morphology

Methods

Collagen assembly is initiated as described in Example 3, and HA isadded to the concentrated collagen solution in the followingconcentrations: 0.5 μg/ml, 2 μg/ml, 4 μg/ml, 8 μg/ml, 20 μg/ml, 40μg/ml, 100 μg/ml, 500 μg/ml, 1000 μg/ml, and 5000 μg/ml. Constructs areassessed on the same timescale as described in Part A above, in parallelwith a negative control (no HA). Bovine HA is obtained from Sigma(#53728, Fluka).

The rate of collagen fibril assembly is determined as described in PartA above, using DIC to analyze the rate of fibril formation (n=3), sTEM(n=1), and immunofluorescence to investigate HA assimilation into (orindependent of) the construct (n=1). Antibodies raised against HA areobtained from Immunology Consultants Laboratory Inc, Newberg, Oreg.(#MHGT-45A-Z).

Results

Increasing the presence of HA increases the rate of fibrillogenesis dueto the physicochemical water sorptive capabilities of the group. HA is aubiquitous ECM carbohydrate polymer that is a prominent feature of loadbearing tissues such as cartilage. HA is transiently expressed insignificant quantities during the early stages of avian development asthe cornea swells and the template-like primary stroma is laid down.Over half the GAG assayed after 50 hrs of chick embryogenesis is HA.

C. A Substitute Hygroscopic Molecule with Potent Co-NonsolvencyProperties Influences the Rate of Type I Collagen Fibrillogenesis andMorphology

Methods

Atelo-collagen monomers are stimulated to assemble (as described inExample 3) in the presence of PEG 400, 600, 1000, 4000, and 8000(NC9063854, AAB2179830, NC9395474, NC9090155, and BP233-100,respectively; Fisher Scientific (Pittsburgh, Pa.)). PEG is added usingthe optimum concentrations determined in Parts A and B above, andmonitored on the same timescale, along with a negative control (n=2 foreach concentration).

Constructs are assessed as outlined in Part B, above. Anti-PEGantibodies are obtained from Epitomics, Burlingame, Calif. (#2061-1).

Results

The substitution of GAG for a similarly hygroscopic syntheticmacromolecule increases the rate of fibrillogenesis throughphysicochemical water sorption. PEG is a simple hydrophilicnon-interacting polymer that can mimic the co-nonsolvency effect ofsulfated GAG, which modulates the rate of collagen fibrillogenesis. Avariety of comparatively small molecular weight PEG are used toapproximate the fixed charge density provided by GAG in biologicalsystems such as cornea, cartilage and intervertebral disc, and theresulting rate of fibrillogenesis is monitored. The rate of fibrilassembly is expedited in comparison to control samples in the absence ofHA/GAG and PEG. DIC images taken at specific timepoints are assessed tomonitor the rate of fibril assembly between sulfated and non-sulfatedGAG chain inclusion against comparable controls. These results determinethe optimum conditions for controlled collagen precipitation.

D. PG Core Protein and Collagen Interaction Affect Fibril Banding andDiameter

The presence of either lumican or decorin core proteins nucleatesself-assembling collagen fibrils to aid ordered banding and diametercontrol, and the increased order of assembly can inhibit the rate offibrillogenesis. PG cores perform an interchangeable role in collagenfibril morphology control. Upregulation of lumican in keratocan knockoutmice, and biglycan in decorin null mice both compensate to mask aphenotype. Double knockouts have cloudy corneas comprising abnormallythick collagen fibrils, with both increased fibril diameter and abnormallateral growth.

Methods

Bovine lumican and decorin cores are isolated and purified from freshbovine corneal tissue using a novel method to retain biological activitywith a higher yield than conventional techniques. In this new method,corneas are frozen, pulverized and homogenized. Briefly, PGs areisolated by anion exchange chromatography on Q-Sepharose and treatmentwith chondroitinase ABC or keratanase I/II/endo-β-galactosidase for sidechain removal. Lumican and decorin cores are purified by a secondQ-Sepharose chromatography with affinity chromatographies onheparin—Sepharose and concanavalin A—Sepharose, as described in Brown etal. (Protein Expr. Purif. 25:389-399 (2002)). Core proteins areconcentrated and purified before filtering and storage at −20° C.

Cores are combined with dense collagen mixtures prior to neutralization,concentration and assembly in the quantities outlined in Table 8.

TABLE 8 PG Core Protein Addition Conditions After Concentration Hrs ofassembly Core protein (μg/ml) 0.5 1 6 12 24 36 5 ✓ ✓ ✓ ✓ ✓ ✓ 10 ✓ ✓ ✓ ✓✓ ✓ 50 ✓ ✓ ✓ ✓ ✓ ✓ 100 ✓ ✓ ✓ ✓ ✓ ✓ 200 ✓ ✓ ✓ ✓ ✓ ✓ 500 ✓ ✓ ✓ ✓ ✓ ✓ 1000✓ ✓ ✓ ✓ ✓ ✓

Initial experiments optimize the conditions through variation ofparameters such as equilibrium time to allow core protein binding. Coreprotein additions approximate physiological quantities, with a range ofconcentrations added to result in an excess. Negative controlexperiments are also completed, for direct comparison.

The relationship between collagen fibril spacing and PG cores isinvestigated using DIC to monitor the rate of fibrillogenesis (n=3) andimmunofluorescence to label cores in the whole construct (n=1), asdescribed in Part A above. A closer examination of this relationship isalso completed using immuno-electron microscopy (n=1). Constructs aresubjected to low temperature embedding into lowicryl by automated freezesubstitution (AFS, Leica), followed by core protein labeling using aLumican (L-20) antibody (SC-27718, Santa Cruz Biotechnology, Inc., SantaCruz, Calif.) and 70.6 anti-decorin.

For low temperature embedding, preservation of antigenicity and spatialdistribution of PG cores, PGs and GAGs are achieved using automaticfreeze substitution. Controlling molecules are immunogold labelled(primary antibodies shown in Table 7) as outlined in Young et al.(Invest. Ophthalmol. Vis. Sci. 48:3083-3088 (2007)). Labelled sectionsare viewed on a JEOL JEM-1000 (Tokyo, Japan).

Blocks are sectioned and stained as described in Part A above. Controlexperiments reflect (i) absence of primary antibody, and (ii) absence ofsecondary antibody.

Results

Banding patterns of constructs in the presence of lumican and decorincore proteins approach values seen in healthy ECM, (67 nm axialperiodicity) compared to control samples.

E. Intact PG Affects Cholesteric Self-Assembled Collagen Fibril Bandingand Spacing

Methods

Atelo-collagen are assembled in the presence of intact lumican anddecorin isolated from fresh bovine corneal tissue, as described in PartB above. Pulverized fresh bovine corneas are extracted at 4° C. underguanidine extraction buffer for 48 hrs, 1 ml/100 mg tissue. Extracts arecentrifuged after 24 hrs, the supernatant removed, and fresh buffer usedto resuspend corneal fragments. After successive extractions,supernatants are combined and exhaustively dialyzed against DI.Guanidine extraction buffer is used at pH 5.8-6.8 and includes 4 Mguanidine HCl (Sigma G-4505), 0.05 M NaAc (Sigma S-7545), 0.01 MNa₂EDTA, 0.1 M 6-amino (caproic) acid combined with protease inhibitors(5 mM benzamidine HCl (Sigma B-6506)), and 0.5 mMphenylmethylsulfonylfluoride in methanol immediately prior to use.

For core protein reformation and PG purification, DEAE and affinitychromatography are used as described Dunlevy et al. (Invest. Ophthalmol.Vis. Sci. 45:3849-3856 (2004)). Briefly, lyophilized (Labconc Freezone4.5) extracts are reconstituted in 6.0 M urea containing NaCl, Tris, andCHAPS, and applied to DEAE Sepharose step columns equilibrated in thesame solvent. PGs are eluted from the column, dialyzed against water,and lyophilized. Affinity purification of lumican and decorin isperformed using monoclonal core protein antibodies, removing otherproteins by washing from the affinity substrate in a column format. Coreproteins are eluted from the column, dialyzed against dH₂O, andlyophilized.

PGs are added according to GAG content (assayed against a known sharkcartilage standard by DMMB), reported in vivo. For microquantificationof GAG, a DMMB dye binding assay is used. GAGs bind DMMB reagent vianegatively charged sulfate groups resulting in a metachromatic shift(color change) in the absorbance maxima at 525 nm. Extracts are measuredagainst known dilutions of CS used to generate a standard curve, andconcentration is calculated from the resulting graph. 40 μl sample istested for the presence of sulfated GAG with 200 μl DMMB reagent againstshark cartilage (Sigma C-4384) standards at 525 nm. This assay loseslinearity if GAG concentration is greater than 40 μg/ml, so samples arediluted if needed. Dimethylmethylene Blue reagent is prepared from 16 mg1,9 dimethylemethylene blue (DMMB, SERVA #20335) in 5 ml 98% ethanol, 2ml formic acid, 2 g sodium formate, 1 L with MilliQ H₂O, pH 6.8.

The method is first carried out using the GAG concentrations and methodsdescribed in Part A above (Table 6 supra).

PG additions are then modified as shown in Table 9, where cornealapproximations are shown in white, and tendon in gray. Negative controlexperiments are also completed for direct comparison.

TABLE 9 KS/CS Mixture GAG Addition Conditions for Intact PGs

DIC is used to monitor the rate of fibrillogenesis (n=3). Optionally,QFDE is used to determine construct morphology (n=2) (as described inExample 3), in addition to immuno-electron microscopy (n=1) todemonstrate collagen/PG interaction (as described in Part D, above). GAGchains are labeled using the same monoclononal antibodies described inPart A, above, and 70.6 anti-decorin. X-ray analysis (n=1) offull-thickness, mean center-to-center collagen fibril spacing in fullyhydrated tissue is conducted.

For X-ray analysis, constructs are placed between two pieces of clingfilm to maintain hydration and minimize disturbance through handling,frozen at 253 K, and transported on dry ice to SPring-8 for examinationusing small-angle x-ray scattering. Experiments are conducted onbeamline 40XU using a 30 μm diameter x-ray beam (λ=0.83 Å). Frozenconstructs are thawed under ambient air while wrapped in cling film, andthen secured onto a Mylar sheet and mounted into the x-ray beam pathwith the construct face perpendicular to the incident beam direction.Exposures are obtained from designated points across the entireconstruct, and the resulting SAXS patterns are recorded on a cooled CCDcamera (ORCAII-ER, Hamamatsu Photonics) coupled with an x-ray imageintensifier (V5445P, Hamamatsu Photonics) 3 m behind the specimen.Average x-ray intensity is recorded during data collection by an ionchamber placed between the incident beam and the specimen. X-raypatterns are analyzed using purpose-written, Unix-based softwarefollowed by a graphics and statistics package (Statistica; Statsoft,Tulsa, Okla.) as previously described by Meek et al. (Prog. Retin. EyeRes. 20:95-137 (2001)). In addition to measuring mean fibril to fibrilcentre spacing, the first order equatorial reflection is also used toestimate the degree of local order in the collagen matrix. Averagecollagen fibril diameter is also calculated, as described by Worthingtonet al. (Intl. J. Biol. Macromolecules 7:2-8 (1985)).

Data analysis is carried out in accordance with protocols described byQuantock et al. (Invest. Ophthalmol. Vis. Sci. 42:1750-1756 (2001)),using the position of the first-order equatorial reflection. The firstsubsidiary maximum of the experimental data is further analyzed toascertain average fibril diameter, as described in Meet et al. (Prog.Retin. Eye Res. 20:95-137 (2001)). Constructs are assessed at thespecific timepoints outlined, which are expanded into a binary search.

Results

Collagen interaction with intact lumican and decorin promotes collagenassembly into banded fibrils with altered interfibrillar spacing at arestored rate of fibrillogenesis. Lumican and decorin interact withcollagen via a horseshoe-shaped core protein, with physico-chemicoproperties derived from their GAG content. The increased length andsulfation of CS and dermatan sulfate (DS) carried by decorin, andshorter KS chains on lumican, may be short and long-range mediators offibril spacing.

The D-banding patterns of KSPG/collagen constructs approach closervalues to those seen in cornea, with an increase in fibril order andmore regular fibril diameter and spacing (as measured by x-raydiffraction) compared with controls. Restoration of assembly rate overPG core only assembly rates is observed.

F. Affect of Type V Collagen Co-Assembly on Self-Assembled CollagenFibril Diameter and Spacing

Whether manipulation of Type V collagen concentration enables control offibril diameter in a self-assembling heterotypic collagen mixture (TypesI and V) is evaluated.

Methods

Type V collagen is extracted and purified from bovine cornea in thepresence of protease inhibitors using an acetic acid extraction followedby limited salt fractionation (as described in Example 3). The Type Vcollagen is combined with Type I collagen. Fibrillogenesis is induced asdescribed in Example 3. Type I collagen is combined at variousconcentrations: 1%, 2%, 5%, 10%, 20%, 25%, 30%, and 40%, and the methodsare repeated three times.

DIC microscopy is used to study the rate of fibrillogenesis (n=5). sTEMis undertaken to investigate collagen banding and fibril thickness afterpreservation through standard resin embedding (n=1), and QFDE is used todetermine fibril morphology. To verify Type V incorporation intoheterotypic fibrils (and link fibril diameter effects), immunoelectronmicroscopy is used (n=1), and antibody labeling is carried out afterassembly for a representative number of samples (as described in Birk,J. Cell Sci. 95:649-657 (1990)). Monoclonal antibodies against Types I(5D8-G9/Coll, Abcam, Cambridge, Mass. #ab23446), and V (1E2-E4/Col5,Abcam #ab36382) can be used.

To elucidate the mechanism of ordered matrix assembly in vivo andproduce cross-linked fibrils, selected methods are also conducted usingthe collagen precursor, procollagen. Procollagen terminal domains helpto form the triple helix and inhibit fibril formation until suitable LCconcentrations are reached in vivo, when a wave of protease enzymes arereleased allowing fibrils to rapidly assemble under templatinggeometries in the presence of ancillary molecules. To analyze this, TypeI procollagen is extracted from fetal skin, obtained from Pelfreeze-bio(#57090-1). Procollagen is extracted and purified using the methodsdescribed by Byers et al. (Proc. Natl. Acad. Sci. U.S.A. 72:3009-3013(1975)). Extracts are purified using DEAE-cellulose chromatography.

Type I procollagen is stimulated to assemble using (1) the optimizedtemplating features from Example 3, (2) most favorable PG concentrationfor ordered fibril banding and spacing from Example 5, and (3) theoptimal combination of both. These constructs are subjected to N- andC-terminal domain cleavage (extracted N-Proteinase, C-Proteinase) andevaluated in terms of rate of assembly (DIC), fibril banding and spacing(TEM, QFDE).

Results

Constructs assembled in the presence of Type V collagen show a fibrilbanding and diameter response to Type V collagen manipulation. Thesuccessful manipulation of collagen assembly and morphology by theaddition of collagen controlling molecules enables control of assemblyconditions and constitutes a first generation model of engineeringbiomimetic load-bearing tissue by design. Additionally, information isprovided regarding the optimum conditions for de novo tissue assembly.The ersatz matrix serves as a test bed for strategic knock-out andknock-in experiments, where the roles of auxiliary ECM are identified.

Example 6 Strain-Directed Collagen Degradation in Native Tissue

A. Tensile Degradation Assay Experiments on Bovine Corneal Stroma

The cornea is unique in that it comprises arrays of uniform diameterType I/V heterotypic collagen fibrils arranged in 2 μm thick lamellaethat are parallel to the corneal surface, stacked orthogonal to oneanother and run uninterrupted across the entire cornea. Additionally,the fibrils do not interact with one another covalently and aregenerally thought to “float” in the extracellular matrix. Thus, auniaxially loaded strip of cornea possesses a population of loadedfibrils (aligned with the tensile load) and a control population ofunloaded fibrils (transverse and oblique to the load). To quantify thekinetics of native collagen cleavage in uniaxial strips of cornea, aminiature, thermally-controlled bioreactor capable of applying precisetensile loads/strains to tissue strips in an aqueous environment wasconstructed Church, et al. BMES Oct. 12, 2007). Using this new deviceand more active collagenase (e.g., purified collagenase or MMP), thekinetics of collagen degradation in strips of bovine cornea wereexamined

Methods

Briefly, strips of devitalized bovine cornea were excised, debrided andloaded into the bioreactor. Strips were placed in load control mode withtensile uniaxial applied forces of 0.25 N, 0.1 N or unloaded (aftercreep in) and exposed to 0.05 M bacterial collagenase (crude). Thedetailed methods are described in Example 10, below.

Results

FIGS. 13A-13B are plots of the results that shows strain and area lossas a function of time. FIG. 13 shows degradation curves for nativetissue strip in uniaxial loading bioreactor (load control). The strainversus time curves in FIG. 13A demonstrates that cornea tissue subjectedto lower loads degrades to failure more quickly. In FIG. 13B, estimatesof effective loaded-area loss as a function of time indicate that tissuesubjected to lower loads lose area more rapidly during initialdegradation. Increasing strain reduces rate of area loss (right side ofcurve). (Red—0.25 N; n=5); (Blue—0.1 N; n=6). Control curve showsminimal creep after 15 minutes (n=5). Tissue that was completelyunloaded during degradation lost mechanical integrity faster than eitherloaded sample (n=3).

These figures demonstrate that the rate of area loss (cleavage rate ofcollagen) was significantly faster when the load was reduced (even atthis high concentration of bacterial collagenase). Corneal stripsdegraded more slowly when loaded.

B. Compressive Degradation Assay on Bovine Articular Cartilage

Because collagen with lower tensile load is more susceptible to cleavageby collagenase, compressive mechanical overload of cartilage results inaccelerated collagen catabolism. This has direct relevance toosteoarthritis in that Type II collagen that is relieved of its normaltensile load in cartilage (due to a local compressive overload) may besusceptible to enzymatic cleavage. Thus higher cartilage compressiontranslates to lower collagen tension. When collagen fibrils areprotected by the internal tensile strain generated by the GAG fixednegative charge density, “unloading” the fibrils in cartilageaccelerates collagen degradation (in the presence of enzyme). Collagenwas thus “unloaded” in cartilage plugs (by applied compression) in thepresence of BC and the mechanical properties of the cartilage (stressevolution) were monitored.

Methods

3 mm diameter, bovine tibial plateau cartilage plugs excised from bovineknee were infiltrated with precise microgram quantities of BC bytranspiration. FIGS. 14A-14C show a capillary tube filled with enzymesolution and tipped with a needle inserted into a cartilage plug. Theplug was dehydrated at 4° C., which resulted in the drawing of BCsolution from the capillary tube. The specimen was then rehydrated inPBS at 4° C. and placed in an ELF3100 (Bose, Eden Prairie, Minn.)dynamic mechanical analyzer. This method allowed the precise “rapid”infiltration of a known quantity of enzyme directly into the center of asmall sample volume. Static (unconfined) compressive strains of 10% and20% were applied and the temperature was raised to 37° C. The stressevolution as a function of time was followed for up to three hours. Thedetailed methods are described in Example 10, below.

Results

The compressive modulus of rehydrated cartilage plugs following shambuffer or BC loading was always lower than fresh plugs, and untreatedsamples lost mechanical integrity with time in buffer and while undercompression. This was due to loss of GAGs from the samples. FIG. 15depicts the net loss in compressive strength for samples that were beingdegraded and losing GAG simultaneously. Compared to sham-loadedcontrols, the 20% strain sample first gained strength (circle), thenlost compressive strength slowly, while the 10% strain sample loststrength continuously.

That the 20% strained sample actually gained compressive modulusindicated that loss of collagen (which restrains swelling pressure) wasmore rapid than in the 10% samples.

Example 7 Strain-Directed Collagen Degradation of Reconstituted CollagenGels

An environmentally-controlled microchamber to allow directmicromanipulation and observation of collagen gels was constructed inorder to directly observe the mechanochemical kinetics of collagenfibril degradation by enzymes.

Methods

Microenvironmental Chamber

FIG. 16 depicts the microenvironmental chamber constructed on thesurface of a Bioptechs Delta T4 (Bioptechs, Butler, Pa.) systemcomprising a delta-T dish (0.17 mm, 04200415B), triple plate stageadapter (PN 0402602), Delta T4 culture dish temperature controller (PN0420-4-03) and objective heater controller (PN 0420-4-03). A smallcylinder (height: 2.2 mm, inner diameter 3.5 mm) was affixed to theDelta T4 glass surface. The initial reaction volume was created byplacing 10 μl of collagen solution into the cylinder then covering itwith objective oil (Cargille, non-drying, Type A, viscosity 150, CedarGrove, N.J.) to prevent evaporation. The final reaction volume wascreated by dropping an additional 10 μl of collagenase solution throughthe oil.

Enzyme and Substrate.

Commercially available, pepsin-extracted, bovine, type I, atelocollagenmonomers at a concentration of 3.0 mg/ml was used (PureCol™, INAMED,Fremont, Calif.). Extracted collagen monomers were kept from aggregatingby storage in cold, acidic solution. Commercially available bacterialcollagenase (crude, Sigma-Aldrich C0130, lot 016K1251) was used.

Optical Imaging.

Differential interference contrast (DIC) or Nomarski microscopy is abeam-shearing interference method that detects gradients in the index ofrefraction (optical path). Reconstituted type I collagen fibrils wereresolved by DIC. DIC optics were mounted to a Nikon TE2000E (Nikon,Tokyo, Japan) inverted microscope equipped with a 60×, 1.45 NA PlanApochromat objective and a digital camera (CoolSNAPHQ2 1394,Photometric, Pleasanton, Calif.). The microscope was also outfitted witha Perfect Focus® interference feedback stage controller capable ofcontrolling vertical drift to less than 50 nm (Nikon, Tokyo, Japan).

Formation and Micromanipulation of Reconstituted Collagen Micronetworks.

To generate micronetwork strain, a pair of micropipettes were fitted toEppendorf TransferMan® NK2 micromanipulators (Eppendorf, North America)and were mounted on the stage (H101A, Proscantm II, PRIOR Scientific,Rockland, Mass.) of the Nikon TE2000E. Prior to mounting, the tips ofthe pipettes (fire polished borosilicate with filament, OD 1.0 mm, ID0.50 mm) were custom manufactured for minimum taper using a P-97Flamming/Brown micropipette puller (Sutter Instrument Company, Novato,Calif.) with a five step program setting (Heat=712, pull=40, Vel=15,Time=150). To facilitate binding of collagen to the pipette tips, theywere first plasma-cleaned (PDC-32G, Harrick Plasma, Ithaca, N.Y.) for0.5 minutes at 50 watts and 200 mTorr. The pipettes were thenfunctionalized by silanization (3 mercaptopropyl-trimethoxysilane)followed by N-(γ-Maleimidobutyryloxy)succynimide ester (GMBS) treatment.The functionalized micropipettes were positioned via themicromanipulator such that they could be observed in close proximity(about 20 μm apart) at 600×. Collagen monomers were prepared forfibrillogenesis according to the packet insert (by gentle mixing ofbuffer (PBS 10×), NaOH (0.1 M) and the collagen solution (3.0 mg/ml) inparts of 8:1:1). To produce a fibrillar network or gel, 10 μl ofneutralized collagen in buffer was added to the Delta TPG dish, coveredwith the immersion oil, and the temperature was raised to 37° C. Asparse gel micronetwork formed both around and between the pipettes. Themicromanipulators were then adjusted to produce a network strain of upto 50% based on initial pipette separation.

Collagen Network Degradation.

10 μl of 0.025 M bacterial collagenase in DMEM was dropped through theoil immediately following micropipette repositioning to permitinteraction with the strained micronetwork. In this way, the timing ofcollagenase addition was controlled to account for stress-relaxation.The final concentration of enzyme-to-monomer resulted in a 3.125:1ratio, allowing the presence of more enzyme molecules than availablecollagen monomers at all times in the chamber.

To analyze any systematic effect of enzyme diffusion delay on thecalculated degradation rates, experiments were visually screened forunequal degradation onsets in the unstrained fibrils on either side ofthe strained fibrils. Further, a conservative set of statisticalcomparisons between the calculated degradation rates of the unstrainedand strained fibrils were carried out to normalize for delay in theonset of degradation of the strained fibrils. The timing of collagenaseaddition was controlled by consistent addition immediately followingpipette repositioning.

Image Signal Processing.

The DIC images extracted during the experiment were subjected to regionof interest (ROI)-based edge detection for quantifying collagendegradation rates. To reduce intensity variations across a large fieldof view, smaller regions of interest were chosen for analysis tominimize the effect of spatial contrast variation on the edge detectionalgorithm. The contrast for each strained and unstrained ROI wasadjusted to a fixed width gamma line in MATLAB (The Math Works, Inc.,Natick, Mass.) to normalize fibril edge sharpness across time. Theinitial gamma line width was determined by the first image taken in theROI and held constant for the remaining data in the image sequence. Thecontrast normalized images were then smoothed using a Gaussian filter bya small value (R=1) to reduce the background noise. Edge detection wasperformed on the temporal stack of strained and unstrained ROI imagesusing a Canny-Deriche edge detection algorithm (alpha=0.5) in ImageJsoftware (NIH, Bethesda, Md.). The edge-detected 32 bit grayscale imageoutput was converted to 8 bit, and the background noise (correspondingto fibril-free areas within each ROI) was subtracted using MATLAB. Thesummation of all of the pixel intensities (which reflect DIC gradientstrength and thus diameter) in an image was assumed to represent theintegration of fibril length (detected-edge length)×fibril diameter(gradient intensity) and thus represents a measure of the remainingfibril volume. Summation values for each ROI were normalized topercentages by dividing all values by the maximum value obtained forthat ROI during the run. Thus the values obtained (which wereinterpreted as the percentage of remaining fibril volume in the 2-Dslice), were plotted against time for both the strained and unstrainedregions. The resulting plots tracked the rate of fibril volume loss dueto enzymatic degradation of each of the collagen fibril populations inthe ROIs.

Edge Detection Method Validation.

To validate the edge-intensity image processing method used to determinerelative fibrillogenesis and degradation rates qualitatively, an idealnetwork of constant-diameter fibrils was simulated for a range ofdiameters and then evaluated using the same image processing method.Collagen fibrils are modeled as infinite cylinders in the plane ofobservation where the light scattering effects can be calculated bysolving the cylindrical wave equation as shown by Bohren & Huffman,Absorption and scattering of light by small particles, New York:Wiley(1983). While the orientation of fibrils in reconstituted networks israndom, DIC microscopy has excellent out of plane rejection and thusin-plane fibrils will dominate the intensity data. Forward scatterintensity profiles can be generated for a range of fibril diameters andin-plane angles with respect to incident light polarization, θ_(P). Aconstant-diameter fibril network was simulated by randomly superimposinggenerated profiles for different values of θP with noise added. The DICimage of this simulated network was calculated to produce the finalexpected image.

Quick Freeze Deep Etch (QFDE) Imaging.

Morphology preserving QFDE imaging was used to evaluate the initialpolymerized condition of the fibrillar networks. Formed collagen gelswere slam frozen at −196° C. on copper blocks with a portable cryogen(Delaware Diamond Knives, Wilmingdale, Del.). The samples weretransferred under liquid nitrogen into a custom CFE-40freeze-fracture/freeze-etch device (Cressington, Watford, UK). They werethen etched at 95° C. for 45 min and rotary shadowed at −130° C. with 2nm platinum and carbon at 20° angle and backed by 90° angle carbon.Replicas were picked up on grids and imaged digitally at 70 kV on a JeolJEM-1000 (JEOL, Tokyo, Japan).

Data Analyses and Statistics.

Degradation times within specified ROIs were extracted from the curvesproduced by the image processing algorithm described above. The measured“degradation” time was defined as the time interval (ΔT) between 95%(I_(start)) and 10% (I_(finish)) of I_(max) (FIG. 17). The lower boundwas chosen to be 10% of I_(max), as there were multiple strained fibrildigestion experiments that did not reach 5% of (I_(max)) prior to theend of the experiment. Degradation times from experimental runs wereanalyzed for Normality (Shapiro-Wilks test), skewness and kurtosis, andoutliers removed using SPSS15. A paired, two-tailed Students T-test(Excel, Microsoft Corp.) was used to assess statistical significance(p≦0.05) in the processed data sets (n=10) and control experiments(n=2).

Results

FIG. 18 demonstrates that reconstituted collagen fibrils strainedbetween two micropipettes degraded at a slower pace than unloadedcontrol fibrils in the same gel. The access of the enzyme to thestrained fibrils was not limited via strain-induced diffusioncoefficient reduction as indicated by the loss of dense fibrils thatwere compressed under the bottom pipette (arrows). With bacterialcollagenase, eventually all fibrils were lost to enzymatic cleavage.However, a statistically significant difference in the observed time todegradation (signal loss) between unloaded and loaded fibrils wasdetected, p=0.00129.

In these experiments, it was possible to discern that strained fibrilssurvived the enzymatic attack longer than unstrained fibrils. Thevideo-enhanced and edge-detected DIC elucidated the survivingstructures. Initial evaluation of simulated edge intensities using theimage processing method described above showed a linear relationshipbetween fibril diameter and DIC edge intensity for fibril diameters of60-250 nm. Within this range, edge intensity provided a goodqualitative, and potentially quantitative, method for evaluation ofrelative fibril diameter. Outside of this range, the edge intensity didnot seem to vary much with diameter.

FIG. 19 provides a temporal plot of the integration of detected edgestrength, which was interpreted as being directly reflective of fibrilvolume, from two ROIs (strained or unstrained fibrils). Analysis of thedata demonstrated that normalized degradation times for unstrained andstrained fibrils were statistically different. Moreover, in everyexperiment, degradation was consistent: ΔT_(S)>ΔT_(U). Quantitatively,the loss of edge detection by DIC between I_(start) and I_(finish) instrained and unstrained fibrils respectively, mean degradation time(±SE) ΔT_(S)=1113±260 secs, and ΔT_(U)=654±149 secs (p=0.0071). Thissignificant difference included an allowance for the “delay” in theonset of degradation that could be attributed to diffusion differencesacross the experimental domain. However, the delay in the onset ofdegradation was consistent and only affected the strained fibrils. Theeffect of strain was efficiently isolated by the presence of control(unstrained) fibrils microns away from the strained ROI, therefore thereproducibility of the initial gel state was unlikely skewing theanalysis.

Comparison of the average “onset” time for degradation (defined sd 95%of I_(max)); mean (±SE) for strained fibrils was T_(Sstart)=723±357secs, and for unstrained fibrils was T_(Ustart)=586±323 secs. T_(finish)time also increased under strain; with a mean (±SE) of 1836±587 secscompared to an unstrained mean (±SE) of 1240±454 secs.

The total loss of strained fibrils was also compared during the time ittook for unstrained fibrils to fully degrade as defined by I_(Ustart)and I_(Ufinish) (a total of 85% edge detection loss). The loss ofstrained fibrils was significantly smaller during this period, with amean fibril loss of (±SE) 63±4%, p=0.0002. When fibril degradation wasdefined to begin when the unstrained fibrils reached 95% of I_(max)(I_(USstart)) and when degradation was defined to end when each set offibrils reached 5% of I_(max) (I_(SFinish)), the mean (±SE) ΔT_(S) was1250±306 secs, and the mean ΔT_(U) was 654±149 secs, indicating ahighly-significant difference in degradation time (p=0.0078). In controlexperiments where fibrils in the reconstituted matrix (including thosebetween the functionalized pipette tips) were unstrained, the mean (±SE)digestion time ΔT (between pipette tips) was 510±180 secs, and in thesurrounding matrix ΔT_(U) was 565±215 secs, which was a non-significantdifference (p=0.5250). This implies that strain was attributed as thecausative factor in degradation time differences.

MMP-8 was tested to determine if the strain-induced resistance ofcollagen to BC extended to this mammalian collagenase. FIGS. 20A-20Cdemonstrate that MMP-8 preferentially removed fibrils that were notunder clear mechanical strain. The pipettes moved apart during theexperiment due to loss of fibrils that had polymerized and tethered thepipette to the matrix. These fibrils are out of view of the images inFIGS. 20A-20C). In most cases, fibrils under load were retained for theduration of the experiment (unlike bacterial collagenase). MMPs are moresensitive to strain-induced molecular distortion given their specificityfor one cleavage location and the reorientation of collagen monomerduring cleavage.

Example 8 Strain-Directed Collagen Degradation and Monomer Incorporationin Reconstituted Collagen Gels

To determine whether strain preferentially protects loaded fibrils andalso enhances their reinforcement and growth, monomers were assayed forincorporation into fibrils under strain.

Methods

Type I Collagen was polymerized around functionalized micropipettes asdescribed in Part A above; however, during the polymerization process,the micropipettes were slowly moved apart (at a rate of about 1 μm/min-2μm/min)

Results

FIGS. 21A-21D are DIC image sequences that demonstrate the preferentialincorporation of monomers into a loaded cluster of fibrils between twopipettes that were moving apart. The initial collagen connecting thepipettes appeared to both thicken and extend in length. In thebackground, a random network of fibrils is shown gelling. This suggeststhat collagen monomers incorporate into fibrillar structures that areunder strain. Further evidence is depicted in FIG. 20A, where fibrilsthat formed between two pipettes and between the left pipette and thegel were “strained” early in the polymerization process. The fibrilsappeared “thicker” and the fibrillar structure was denser thanbackground fibrils that formed with no load. Remodeling and growth ofcollagenous, load-bearing ECM can be accomplished efficiently andwithout direct cell intervention by the addition of monomer by collagenfibrils when loaded.

Example 9 Statistical Reaction-Diffusion-Strain Model of CollagenCatabolism

The kinetics of collagen catabolism by bacterial collagenase or humanskin fibroblast collagenase follows Michaelis-Menten rate kinetics. Whenincorporated into fibrils, monomer availability is reduced due to fibrilinsolubility and steric hindrance leaving only surface monomersavailable for degradation. To incorporate strain-dependence into thereaction rate coefficients and to model the viscoelastic strain-behaviorof loaded, degrading corneal strips, a 1-dimensional reaction-diffusionmodel was extended.

Methods

Both the forward binding rate coefficient and cleavage rate coefficientwere assumed to possess simple bounded, linear strain-dependence (seeFIG. 22). Since the kinetic parameters, K_(cat) and K_(m), forClostridium histolyticum collagenase acting on Types I and III collagensare similar in magnitude to those of human enzymes acting on theirpreferred substrates, the data from Example 6 were qualitatively “fit”to the model.

Results

FIG. 22 shows a family of curves produced when the binding rate andcatalysis rate coefficients were modeled as linear functions of strain.The data qualitatively matched that depicted in FIG. 13 (where theenzyme binding decreased with strain, but the cleavage rate increasedwith strain) and included a good fit of data from 0.5 N load controlexperiments.

To achieve the characteristic family of curves, the binding ratecoefficient was forced to decrease with strain, and the cutting ratecoefficient was forced to increase with strain. The net effect produceda “sweet” spot in the strain/degradation behavior of corneal collagen(about 4%), which can be maintained to protect a population of collagenfibrils. Strain-induced decreases in enzyme diffusion rates produced asimilar set of curves. The strain-induced decrease in diffusion is about54% for a change in strain of only 3%.

Example 10 Determination of the Degradation Kinetics of Whole Tissues

Collagen fibril survival is a function of the strain energy density andthe concentration of catabolic enzymes present in the matrix. Nativetissues, whose resident collagen fibrils are placed under varyingstrains, are exposed to catabolic enzymes (e.g., MMP-8, MMP-13,Cathepsin-K, and BC) and the relationship between strain and enzymereaction rate kinetics is determined

A. Relationship Between Strain and Enzyme Reaction Rate Kinetics inNative Tissue

A combination of applied load/strain and enzyme concentration “protects”loaded fibrils from removal (but permits cleavage of unloaded fibrils).A uniform force is applied on one set of collagen fibrils from cornealstroma while another set remains unloaded. Corneal transparency allowsreal-time observation of degradation of off-axis fibril arrays viacross-polarizers. MMP-8 and BC are the enzymes for the cornea's Type I/Vheterotypic collagen fibrils.

1. Titration of MMP and BC Against Applied Fixed Load

Combinations of load and enzyme concentration are established thatremove unloaded fibrils.

Methods

Uniaxial tensile specimens are prepared from fresh bovine eyes (40-100lbs, Research 87Inc. Boylston, Mass.). Corneas with a surroundingscleral rim are dissected away from the globe and debrided prior to celllysis by multiple freeze-thaw cycles. Freeze-thawing has little effecton corneal ultrastructure. A central corneal strip withinferior-superior orientation is extracted (0.7 mm thick×17.5±2.5 mmlength×6 mm wide) and inserted into a custom-built bioreactor chamber,as described in Church, et al. BMES Meeting, Oct. 12, 2007. The scleralrim is fastened into custom grips. Silicon adhesive is used to protectattachment points from digestion. The chamber is filled with pre-heatedmedia (DMEM with 1.0% gentamycin) and a small preload of 0.01 N isapplied to produce an effective “zero” strain. Following a short“creep-in” period, activated MMP-8 (Chondrex Inc.—5001, Redmond, Wash.)or BC solution is used to replace the loading media.

MMP's are stored in Tris Buffer, pH 7.6, containing 0.05 M Tris, 0.2 MNaCl and 5 mM CaCl₂. MMPs are activated with p-AminoPhenyl MercuricAcetate (APMA) at a 20:1 ratio and incubated at 37° C. for 1 hr beforeinjection into the chamber.

A binary search is used to titrate a range of enzyme concentrations(beginning with a concentration equal to 1/10th the available collagenmonomer in the corneal strip). Strips are held in load-control mode withapplied forces representing sub-physiological, physiological andoverload (0.1 N, 0.25 N, and 0.5 N) in the presence of the active enzymeat 37° C. Strain is recorded. Polarization images are taken every 30 secfor the duration of the experiment with the cross polarizers aligned at90-0° or 135-45° to the load axis. Control experiments are conductedwith heat-deactivated enzyme. Significant deviation of the strain fromaverage control strains at 3 hr results in a binary reduction in thetiter concentration. Stable strain is defined as a deviation fromcontrol strains of ±2.0% after 3 hrs. Production of a “stable-strain”results in a binary increase in titer concentration. Once the titrationthreshold concentration is established, the assay is repeated 5 timesand assessed for preferential loss of unloaded collagen as describedbelow.

Polarizing Light Microscopy (PLM) is used to qualitatively observecollagen loss. Specimens are imaged (Prosilica-CV-640, Burnaby, Canada)through crossed-polarizers. Polarization images at the center of thesample are taken using uniform illumination with the load axis eitheraligned with one of the cross-polarizing lens axes or at a 45° angle toboth lens axes. Images are examined qualitatively and quantitatively forloss or gain of birefringent signal intensity.

Briefly, the unique lamellar structure of central corneal fibrils isilluminated by passing polarized light through the sample. Alignment ofone polarization axis with the specimen axis (load direction)illuminates off-load-axis (unloaded) fibrillar arrays. Loss ofbirefringence signal indicates loss of unloaded fibrils.Immunofluorescence is used to analyze Type I collagen loss across theentire sample in comparison to control samples, using a collagen Ispecific antibody (Abcam, Cambridge, Mass.) (n=2). TEM is used toexamine this relationship on the fibrillar scale in a representativenumber of samples.

For TEM analysis, specimens from the tensile loading chamber are fixedwhile held to uniform thickness between coverslips. The center of eachspecimen is imaged at low magnification (3000×) both transverse to andin parallel with the applied load. The number of collagen fibrils in 5random images oriented perpendicular to the load axis are counted. Thenumber of perpendicular (not load bearing) fibrils are compared acrossdigested and non-digested specimens.

Collagen loss into the media is also measured by a hydroxyproline assay.Released fibrils are hydrolyzed under 1 ml/mg construct 11.7 N HCl at110° C. overnight, and then freeze dried to remove acid. Driedhydrolysates are reconstituted in DI before centrifugation to removeparticulate material. Hydroxyproline residues are assayed in 30 mltriplicate against known standard dilutions of 0 mg/ml, 2 mg/ml, 4mg/ml, 6 mg/ml, 8 mg/ml, and 10 mg/ml, with 70 ml diluent, 50 mloxidant, and 125 ml color reagent, and read at 540 nm after 10-20 minsincubation at 70° C. Hydroxyproline content in the unknown constructsare calculated from the standard curve on each 96 well plate. Collagencontent is extrapolated by multiplying hydroxyproline content by 7. (500ml Stock buffer: 28.5 g Sodium acetate trihydrate, 18.75 g Tri sodiumcitrate dehydrate, 2.75 g Citric acid, 200 ml Propan-2-ol. Diluent: 100ml Propan-2-ol, 50 ml distilled water. Oxidant: 0.7 g Chloramine T, 10ml distilled water, 50 ml stock buffer. Color reagent: 7.5 gdimethylamino benzaldehyde, 9.64 ml Perchloric acid 70%, 1.61 mldistilled water (=60% acid), 62.5 ml propan-2-ol. Hydroxyproline stocksolution: 10 mg purified hydroxyproline (Sigma) in 10 ml DI=1 μg/μlstock solution).

Extraction of enzymatic cleavage rate coefficients is performed asdescribed in Part 4, below.

2. Titration of MMP and BC Against Applied Fixed Strain to Determine theThreshold Concentrations that Yield Constant Load

Combinations of strain and enzyme concentration are established thatremove unloaded fibrils. Strain control is less dynamic and providesmore stable data at each strain than load control.

Methods

Corneas are prepared and fitted into the chamber as described in Part 1,above. Strains are applied at about 2%, 3%, 4% or 5%. All specimens areassessed for morphological and biomechanical changes as described inPart 1 above using PLM, sTEM, and immunofluorescence. Extraction ofenzymatic cleavage rate coefficients are performed as described in Part4, below.

3. Effect of Cyclic Strain on the Rate of MMP and BC Cleavage

Dynamic strains of appropriate frequency alter effective enzyme reactionrate kinetics by modulating binding affinity and cutting efficiency.Examination of the collagen cleavage enzyme reaction equations revealsthree reaction rate coefficients, forward binding of enzyme to monomerk_(m), unbinding k_(u) and cleavage k_(cat). These rates possessdifferent time scales (particularly due to the asymmetric nature of theforward and reverse binding coefficients time constants), indicatingthat cyclic loading produces marked accelerations or decelerations incollagen catabolism. Extracted enzyme reaction rates are used todetermine the actual values of frequency and dynamic strain that providemaximum protection of loaded fibrils (see Part 4, below).

Methods

Corneal test specimens are prepared and loaded into the chamber asdescribed in Part 1, above. The specimens are then subjected to dynamicstrains (about ±1% and ±2%) around a fixed static strain (about 3% and4%) and at multiple frequencies (about 0.5 Hz, 1.0 Hz, and 2.0 Hz).Samples are strained at static strain until the stress-relaxationtransient dissipates (about 30 min). Dynamic strains are superimposedonto the static strain for a period 10 hrs in the presence of active(experimental) or inactive (control) enzyme. Force is recorded duringthe entire procedure. Corneal strips are assessed as described in Part1, above, for morphological and biomechanical changes before, during andafter enzyme degradation (using PLM, sTEM, and immunofluorescence).Strain and load information obtained is reported as Strain vs. Time,Strain Rate vs. Time, and Strain Rate vs. Strain Percentage in loadcontrol tests. For strain control tests, Load vs. Time and Load Rate vs.Time are reported. Collagen loss into the media is also measured byhydroxyproline assay at intervals that depend on enzyme activity. Oneprocedure for which the titration produces good results (demonstrableloss of unloaded fibrils) is repeated with high-angle beamline. Collagenmass changes are assessed and collagen organizational changes duringenzyme exposure are directly quantified by X-ray diffraction.

To measure fibril orientation using high angle X-ray diffraction, eachcornea is mounted into a modified Perspex experimental chamber andexposed to the loading/enzyme conditions outlined in Parts 1 and 2,above. Media is removed to obtain a scatter pattern, and is replacedimmediately after x-ray exposure (about 30 secs). A beam of parallelX-rays is scattered by an array of collagen fibrils perpendicular to thedirection of the incident X-ray beam. At small angles, the equatorialX-ray scattering arises from the distribution of collagen fibrils, andat wide angles it arises from the distribution of collagen moleculeswithin the fibrils arranged approximately parallel to the fibril axis.Irradiation is performed using a 0.1 mm cross section beam (Beamline122, camera length 12 cm). Scattering patterns are collected on an imageplate detector (Mar Research, Hamburg, Germany) at each point across a25 point grid in the center of the corneal strip. Scattering fromnoncollagenous components of the tissue is subtracted, and the patternsnormalized to account for fluctuations in x-ray beam intensity.

4. Extraction of Enzyme Reaction Rate Coefficients Using StatisticalReaction-Diffusion-Strain Model of Collagen Catabolism

Methods

A Reaction-Diffusion-Strain model of collagen degradation is used to fitthe model output to data for both load-control and strain-control.Degradation of collagen (Types I-V) by bacterial collagenase and humanskin fibroblast collagenase follow Michaelis-Menten rate kinetics.Insoluble fibrillar array degradation in a gel has been modeled using acoupled set of reaction-diffusion equations (see Tzafriri et al.,Biophys. J. 83:776-793 (2002)). This model is modified for loadedcorneal strip degradation. The geometric monomer availability constant,k, is changed in the following equation, where a_(m) and d_(f) are thecorneal monomer intermolecular Bragg spacing and the fibril diameter,respectively.

$k = {4\left( \frac{a_{m}\left( {r,0} \right)}{d_{f}\left( {r,0} \right)} \right)\rho_{o}^{1/2}}$

Collagen in a GAG matrix is modeled as a viscoelastic material with atime dependent modulus approximated using the standard linear model(SLM) and the following equation.

${{E(t)} = \frac{E_{1}}{1 - {\frac{E_{2}}{E_{1} + E_{2}}{\exp \left( {{- t}/\tau} \right)}}}},{\tau = {\eta_{1}\frac{E_{1} + E_{2}}{E_{1}E_{2}}}}$

For increased accuracy, E₂ in the SLM is replaced by 1 nested SLM andreproduces the strain response of a 0.25 N uniaxially loaded cornealstrip. The viscoelastic parameters E1, E2, E3, η1 and η2 are extractedfrom the normalized load-control experimental data using a boundedLevenberg damped least squares method (see Bard, Nonlinear parameterestimation. 1973, New York; Academic Press).

To reproduce the loss of load-bearing fibrils in degrading tissue, theequilibrium modulus in the SLM is assumed to be proportional to thenumber of loaded monomers.

The model is discretized, the spatial second derivative governingdiffusion is approximated using the centered difference method, and thesystem is integrated numerically.

Where s is the variable vector (concentrations of enzyme, monomer,enzyme-bound monomer, and degradation products), θ is the parametervector (initial degradation rate constants, initial diffusioncoefficients and strain-dependence parameters affecting both rateconstants and diffusion coefficients), and h is the operator vectordefined by the initial equations derived by Tzafriri et al, Biophys. J.83:776-793 (2002). Differentiating the following equation with respectto θ:

yields the following set of differential equations in δs/δθ:

${\frac{}{t}\left( \frac{\partial s}{\partial\theta} \right)} = {\frac{\partial h}{\partial\theta} + {\frac{\partial h}{\partial s}\frac{\partial s}{\partial\theta}}}$

This set is numerically integrated simultaneously with the original setfrom Tzafriri over time and space to obtain the sensitivity matrixJ_(t,I). From here the model is fitted to data obtained herein using abounded Levenberg damped least squares method.

The principal adjustable parameters for the fit are the binding andcleavage rate coefficients. The oscillatory strain experiments describedin Part 3, above, are run at frequencies and strains based on relevanttime-scales (for diffusion and binding) extracted from the model.

To model corneal tissue, the tensile loading experiments are modeled asa 1-D problem across the corneal thickness assuming uniformity over thewidth and length of the tissue strip. The concentration of availablemonomer is calculated using fibrillar and molecular spacing data alongwith a geometrical correction. The strain distribution in the modelcornea is based on statistical values of collagen fibril alignmentderived from experimental data.

The diffusion coefficient of collagenase and monomer in the cornea isobtained by comparison of compiled diffusion coefficients for similarsize molecules and Stokes radii. Gelman et al. (J. Biol. Chem.255:8098-8102 (1980)) provide the diffusion coefficient of monomer insolution (0.78×10⁻⁷ cm²/s) as well as in forming RCNs (monomerconcentration 0.1 mg/ml, 0.15×10⁻⁷ cm²/s).

The bovine cornea comprises Type I/V heterotypic fibrils but ispredominantly Type I collagen. Thus, collagen lysis rates in the modelare approximated using known Type I collagen degradation data. Fordegradation of Type I collagen by MMP-1, K_(m) is 0.8 μM and thecatalysis rate constant, K_(cat), is 34.2 h⁻¹. K_(m) and K_(cat) for thedegradation of Type I collagen by Clostridium histolyticum collagenase(CHC) are 3.1-5.5 μM and 900-2100 h⁻¹, respectively. These values areused as initial values with which to seed the model.

If a single family of parameters (initial enzyme rate constants andenzyme rate constant strain-dependencies) predicts values in agreementwith experimental data for a specific setup and load function, then themodel is valid for that load function.

Results

The model fits experimental data for values of reaction-ratecoefficients that depend on strain either linearly or with a low orderfunction. The forward binding coefficient, K_(m), decreases linearly asa function of strain, while the cleavage rate constant, K_(cat),increases linearly with strain.

A clear, consistent relationship between strain and the reaction ratecoefficients is obtained. A “target” strain (as a function of enzymeconcentration) is identified that can protect loaded fibrils whileallowing enzymes to remove unloaded ones. Such a relationship is used inthe engineering of tissue.

B. The Relationship between Strain and MMP Reaction Rate Kinetics inNative Tissue

Increasing compressive load on cartilage leads to increased collagencatalysis by MMP-13 and Cathepsin K. MMPs and Cathepsins play a role inthe degradation of cartilage in osteoarthritis. Unloading collagen canenhance catabolism via enzymes and may be involved in the initiation andprogressive nature of the disease. Chronic local compressive overloadingof cartilage can lead to chronic tensile unloading of the internalcollagen network, making the network susceptible to cleavage byavailable MMPs or Cathepsin K.

1. Titration of the Concentration of MMP-13 and Cathepsin K Against

Equilibrium Hydration in Cartilage Plugs Under Static Osmotic Stress

Methods

Cartilage samples (6 mm diameter, 4 mm length) are excised from thesuperficial and middle regions of bovine tibia articular cartilage andtranspiration-loaded with MMP-13 or Cathepsin K (in activation buffer)for 12 hrs at 4° C. Enzyme loaded samples are rehydrated in 0.15 M NaClcontaining 300 mM L-threose (Sigma, St. Louis, Mo.), 5 units/mlpenicillin (Sigma), and 5 μg/ml streptomycin (Sigma) for 24 hrs at 4° C.before enclosing inside dialysis tubing (1000 Da, Spectrum).MMP-13/Cathepsin K thermosensitive activation is induced by incubationat 37° C. 50 ml-80 ml calibrated, osmotically active PEG-20 kD (Fluka,Buchs, Switzerland) solutions are used to exert osmotic stress on thecartilage plugs as described by Basser et al., Arch. Biochem. Biophys.351:207-219 (1998) and Verzijl et al., Arthritis Rheum. 46:114-123(2002). The tensile stress on the collagen network is calculated fromthe “balance of forces” at equilibrium hydration, as described in Basseret al., Arch. Biochem. Biophys. 351:207-219 (1998). For each PEGconcentration, MMP-13 concentration (beginning at 0.1× collagen monomerconcentration in cartilage) is incrementally decreased using a binarysearch, to find the concentration at which the equilibrium hydration isno different from controls (indicating total collagen fibril protectionby internal tensile stress). The experiment is run four times. Controlexperiments are conducted using activation buffer containing heatdenatured MMP-13/Cathepsin.

Equilibrium hydration is plotted as a function of enzyme concentrationand internal tensile load on the collagen as defined in Basser et al.,Arch. Biochem. Biophys. 351:207-219 (1998). Digested explants areprocessed as described in Part A, above, for sTEM and immunofluorescencewith SPM239 against collagen II (Abcam, Cambridge, Mass.).

Results

An inverse relationship between internal collagen stress and titratedMMP/Cathepsin K concentration (and thus a directly proportionalrelationship between compressive stress and collagen catabolic rate)indicates that compressive overload of cartilage, which leads to lowerinternal collagen tensile stress, can lead to collagenolysis.

2. Titration of the Concentration of MMP-13 and Cathepsin K Against

Equilibrium Hydration in Cartilage Plugs Under Cyclic Strain

Methods

The protocol described in Example 6 is performed with MMP-13/Cathepsin Kand with (6 mm diameter×4 mm length) cartilage plugs. Briefly, enzymeloaded cartilage explants (removed and transpiration-loaded as describedin Part 1, above) are rehydrated in DMEM for 5 hrs at 4° C. Enzymethermosensitive activation is induced at 37° C. in the controlledenvironment of a compression bioreactor (Bose/Enduratec ELF 3100 DynamicMechanical Analyzer, Bose Corp., ElectroForce Systems Group, EdenPrairie, Minn.). The compressive piston assembly is preciselymanipulated using LinTalk software to determine “zero” strain and theapplied DC offset strain is set to 10%, 20%, or 30%. Cyclic strainamplitudes of ±2% and ±4% are applied at frequencies of 0.5 Hz, 1 Hz and2 Hz. Load cell data are monitored continuously via the LabVIEWinterface during the degradation. After 3 hr, the samples are returnedto “zero” initial strain while load is monitored for one hour. A similarenzyme titration is used to find the concentration of MMP for which theload at “zero” initial strain does not rise above “zero,” indicatinglittle or no collagen catabolism. For this concentration of enzyme, theprocedure is repeated four times. Control experiments include a seriesof statically-loaded runs and a complete rerun of all “good” experimentswith heat-denatured enzyme.

GAG loss into the surrounding media is quantified using DMMB assay (asdescribed in Example 5), and collagen loss by hydroxyproline assay (asdescribed in Part A, above). sTEM and immunofluorescence are alsocarried out on a representative number of samples, as described in PartA, above.

Results

Changes in the titrated enzyme concentration (compared to staticloading) are detected that depend on both the amplitude and frequency ofthe cyclic strain. A clear repeatable relationship between degradationrate (measured by equilibrium swelling of plugs) and the loading regimeis determined

Example 11 Analysis of the Mechanochemical Kinetics of ReconstitutedCollagen Networks (RCNs) During Degradation and Polymerization

Reconstituted collagen networks resist enzymatic degradation when“strained” and preferentially incorporate free collagen monomer into“loaded” fibrils. Collagen degradation and polymerization depend on theapplied mechanical environment. The effect of mechanical force/strain onthe kinetics of collagen degradation and polymerization in reconstitutedstrained fibrillar networks is quantified.

A. The Kinetics of Collagen Monomer Incorporation/Polymerization UnderMechanical Tensile Load

Load enhances the polymerization rate of collagen self-assembly in thedirection of mechanical tension. Collagen self-assembly ismechanosensitive in vivo, and can therefore be modulated in vitrothrough appropriate loading and monomer concentration. Manipulation ofthese factors enables the determination of the reaction conditions tocontrol fibril diameter and organization/alignment. The dynamicpolymerization of monomers in solution during the application of strainto assembled fibrillar arrays is directly quantified.

1. The Effect of Constant Strain-Rate on the Kinetics of CollagenMonomer Incorporation/Polymerization

Methods

Bovine Type-I collagen is self assembled (as described in Example 7)around micropipettes and equilibrated for 2 hrs. Following applicationof a starting strain (5%, 10%, or 20% of the initial pipette separationof 5 μm) the pipettes are separated at constant rates of 0.0, 0.25, 0.5,0.75 and 1.0 μm/min while FITC labeled bovine Type-I monomer(Arthrogen-CIA®, Chondrex, Inc., Washington) and neutralization bufferare co-injected slowly (at a rate of about 0.025 μl/min) to produce aconstant supply of fresh monomer (see FIG. 23). Concentrations of 0.05,0.1, 0.5, and 1.0 mg/ml FITC labeled collagen are used. Fibrils in thesurrounding media are used as unloaded controls. Monomers can “slide”relative to one another in strained, uncrosslinked RCNs. Local matrixstrain is extracted using embedded microbeads or quantum dots. Eachexperiment is performed 3 times.

Collagen monomer incorporation is examined at intervals and quantifiedusing fluorescence microscopy (FITC excitation λex=490 nm, emissionλem=520 nm). Briefly, corneal strips are oriented horizontally andvertically (for in plane and transverse sections) in a large droplet ofTissue Tek (Miles, Ind., USA), and then processed as described in Younget al. (Invest. Ophthalmol. Vis. Sci. 43:2083-2088 (2007)). A NikonEclipse TE2000-E (Nikon, Tokyo, Japan) microscope is used to observe theconstructs at 25× magnification to achieve full thickness images.

To track fibrils during the entire experiment, low-light-intensity,shuttered DIC imaging is used and combined with fluorescence images toproduce movies of gel remodeling. Fibril ultrastructural morphology isinvestigated using sTEM. For sTEM, collagenous tissue/RCNs are processedroutinely for sTEM as described in Guo et al. (Invest. Ophthalmol. Vis.Sci. 48:4050-4060 (2007)) and are viewed and digitally photographedusing a JEOL JEM-1000 TEM (Tokyo, Japan)

Results

Collagen monomers preferentially incorporate into loaded fibrils. Aquantitative relationship between estimated strain, strain-rate, andmonomer incorporation is produced.

2. Determination of the Effect of Superimposed Cyclic Strain on theKinetics of Collagen Polymerization

Methods

Fibril assembly is initiated as described in Part 1, above. Initialstrains of 5%, 10%, and 20%; strain-rates of 0.0 μm/min, 0.25 μm/min,0.5 μm/min, 0.75 μm/min, and 1.0 μm/min; frequencies of 0.01 Hz, 0.1 Hz,and 1 Hz; and oscillatory strain amplitudes of 0%, 2.5%, and 5% areused. FITC labeled collagen is injected as described in Part 1, above.Fibrils in the surrounding media are used as unloaded controls. Eachexperiment is performed 3 times.

Monomer incorporation is assessed as described in Part 1, above, usingfluorescence microscopy, DIC and sTEM. Fibril monomer incorporation is afunction of static strain, oscillatory strain amplitude and frequency. Aquantitative multiparametric relationship between monomer incorporationand strain parameters is produced.

Results

Preferential assembly of labeled monomers into loaded fibrils isobserved. A consistent repeatable relationship between strain, monomerconcentration and oscillatory parameters is established.

B. The Mechanochemical Kinetics of Enymatic Degradation of ReconstitutedCollagen Networks (RCNs) Exposed to Tensile Strain

Strained-collagen fibrils in RCNs resist enzymatic degradation.Resistance is a function of concentration. The two operative forwardreaction coefficients (enzyme binding and collagen catalysis) are afunction of mechanical strain. Information regarding the rate ofcatalysis is determined using the methods described below by comparingdegradation rates on loaded and unloaded fibrils that are derived fromprocessed optical images.

Methods

RCNs are formed around pipettes as described in Part 1, above. Toprevent monomer sliding in fibrils, some of the RCNs are cross-linked byincubation under 0.1% riboflavin phosphate-20% dextran T 500 solutionfor 1 min before photosensitive activation under UVA irradiation (370nm, 3 mW/cm²) for 30 mins. Media is slowly exchanged with 12 volumes atapproximately 1 μl/min to avoid structural disturbance. Static strainsof 5%, 10%, or 20%, and oscillatory strains of ±2.5%, ±5%, or ±10% areapplied at frequencies of 0.01, 0.1 or 1 Hz to uncrosslinked orcrosslinked gels. Activated MMP-8 or BC containing media is introducedand the kinetics of enzymatic degradation is directly observed. Enzymetitrations begin at 0.1× the concentration of available monomers andexpanded using a binary search, and the threshold concentration isdetermined at which only unloaded fibrils are removed. Experiments atthe final titrated concentrations for each parameter set are repeated 3times.

DIC optical imaging is used to track the rate fibril degradation. Curvesof fibril DIC signal loss were produced by running DIC images through aFourier transform edge detector followed by integration of observededges. The results were qualitatively consistent with observer estimatesof fibril loss rates. TEM is used to examine fibril morphology.Optionally, MMP-8 cleavage is confirmed using SDS-PAGE.

Results

A threshold concentration of enzyme is determined below at which loadedfibrils are not affected. Until that concentration is reached, a clearrelationship between strain, strain-amplitude, frequency and degradationrates is observed for each enzyme. Higher strains lead to lowerdegradation rates for concentrations of enzyme above “threshold”. Thecrosslinked fibrils have an increased resistance to enzymatic attack.

C. “Growing” an Aligned, Fibrillar, Collagenous Array

Connective tissues in vivo form mechanically-stable, aligned structuresthrough a combination of strain-dependent enzymatic degradation andpolymerization. Organized collagenous tissues are produced in vitro byapplying appropriate strain in the presence of excess monomer and anappropriate concentration of enzymes. This “mimics” the growth processof an aligned collagenous structure.

Methods

The chamber described in Part A, above, is modified (see FIG. 24) toallow continuous addition of monomer and enzyme. As shown in the FIG.24, two micropipettes stretch collagen fibrils while an additional twomicropipettes provide a fresh supply of collagen monomers and activatedMMP into the reaction buffer. To accommodate four micropipettes, thechamber volume is increased to 80 μL primarily by increasing thediameter. The initial volume of collagen during polymerization inbetween the pipettes is kept at 20 μL. In the growth phase, the strainregime (rate, frequency and amplitudes), fresh monomer injection (ratesand concentrations), and enzyme concentrations are initially determinedfrom the results.

Growth of the collagen construct is directly observed with live DICmicroscopy. Polarization microscopy is used to assess the alignment ofthe construct periodically during the run. The detailed morphology ofthe structure formed between the pipettes is studied using sTEM.

Results

The preferential removal of unloaded collagen combined with thepreferential incorporation of monomer into loaded fibrils yields aload-adapted, aligned structure capable of “growth”.

Example 12 The Relationship Between Applied Mechanical Load and theBinding Affinity and Cleavage Rate Coefficient of Single Type I CollagenMonomers

Application of a tensile load directly to single Type I collagenmonomers reduces the effective cleavage rate by specific collagenolyticenzymes (bacterial collagenase, cathepsin-K and matrixmetalloproteinases) compared to unloaded controls. This is demonstratedby exposing multiple collagen molecules, in parallel, to degradingenzymes in a custom single molecule magnetic force assay.

Type I human collagen, covalently attached at either end to asuperparamagnetic (SPM) bead and a glass slide surface, is loaded via auniform magnetic field. The time to cleavage is estimated fromprobability distributions of collagen-tethered SPM bead ejection fromthe glass surface following addition of cleavage enzymes. The magneticfield strength is varied to alter the time to cleavage. The relationshipbetween load and effective cleavage rate and the relationship betweenload and the time to complex formation (binding affinity) aredetermined.

A. Quantitative Relationship Between Load and Effective Rate of CollagenMonomer Cleavage for Various Collagenolytic Enzymes

Methods

Commercially available recombinant Type I human collagen (RhC1-003,Fibrogen, San Francisco, Calif.) is tethered between antibodyfunctionalized superparamagnetic beads and a glass surface. For beadpreparation, SPM beads (1.05±0.02 μm diameter, Invitrogen, Carlsbad,Calif.) are functionalized with antibodies to the C-terminus telopeptideof human collagen. The antibodies are attached to the beads via an EDAClinker using the PolyLink Protein Coupling Kit (Polysciences, Inc.,Warrington, Pa.). LF-67 Human αI(1) carboxyl-telopeptide is added toEDAC linker coated beads in solution to allow antibody binding. Theantibody functionalized beads are blocked with BSA and aggregation isminimized by exposure to Triton-X100.

The antibody functionalized beads are then exposed to collagen solutionto promote binding. For glass preparation, 40 mm #1 borosilicate glassslides are plasma cleaned and silanized followed by direct addition ofthe linker GMBS (N-[g-maleimidobutyryloxy]succinimide ester)[GMBS,Pierce, Rockford, Ill.]. LF-116 Human α2(I) amino-telopeptide mixed with10 ml 1% BSA/PBS solution is used to functionalize the glass. Forantibody optimization, antibody dilutions of 1:100, 1:250, 1:500,1:1000, 1:2000 and 1:5000 are tested and optimized, and optionallyexpanded into a binary search. These preliminary experiments areconducted by direct ELISA.

To apply mechanical load, the antibody-coated coverslip is placed into amicroscope-mounted, closed-cell, flow chamber (FCS2, Bioptechs, Butler,Pa.) and exposed to the collagen-coated beads for incubation (1 hr at25° C.). A stack of five neodymium magnets, which are calibrated forfield strength vs. distance using Stoke's drag on a sphere, arepositioned to produce a 10 pN force on the SPM beads. Collagen moleculesreach their contour length of 300 nm at this level of applied load, anduntethered beads are removed. The image plane of the microscope is setto the plane of the centerline of the tethered beads (nominally 800 nmabove glass surface). Activated cleavage enzyme is introduced to thechamber at a rate and concentration that minimizes disturbance, yetprovides rapid transport. Applied loads begin at a level that does notelongate a collagen monomer past its entropic regime (about 2 pN) and isextended into the elastic regime >10 pN. Load is increased depending onthe detected sensitivity of the collagen/enzyme complex. To achieveforces greater than 10 pN, SPM beads of diameter greater than 1 μm areused. Controls are run with heat-inactivated cleavage enzyme. Theseexperiments are repeated to include bacterial collagenase, MMP-1, MMP-8,MMP-13 (all Sigma, Mo.), and cathepsin-K (Biomol International Inc.,PA). At least five different loads per enzyme concentration will be runand three different enzyme concentrations are run per load. Experimentsare repeated three times.

For cleavage events, beads are tracked optically under 40× magnificationon a TE2000 Nikon microscope (Nikon, Tokyo, Japan) using a PhotonicsCoolSNAP black and white CCD camera at one second intervals. Each enzymevs. force experimental run produces population data that relate the timeblocks to the number of ejected beads during that time block. Theposition of the peak of this population curve is the effective cleavagerate. For assessment of stress measurements, though the magnetic stackis calibrated, the dipole moment of each SPM bead can vary. To determinethe applied force with greater accuracy, the Brownian motion of thetethered bead is analyzed. The force on the collagen is then determinedusing the following formula, where z is the average end-to-end extensionlength of the molecule, 300 nm, and δx is the lateral Brownian excursionof the bead center:

$F = \frac{k_{s}{T(z)}}{\left( \left( {\delta \; x} \right)^{2} \right)}$

Z position is determined using the method described in Part B, below.

A relationship is determined between cleavage rate and mechanicalload/concentration for every enzyme that is tested.

Results

Increasing tensile load on collagen monomers increases the time tocleavage.

B. Quantifying the Effect of Tensile Mechanical Load on the Binding RateCoefficient Governing Collagen/Cleavage Enzyme Complex Formation

Methods

Quantum dots (Invitrogen, Carlsbad, Calif.) are prepared according tothe manufacturer's protocol, and bound to GMBS in the same fashion asantibody attachment as described in Part A, above. During a bindingevent, the MMP alters the length, z, of the collagen monomer or altersthe Brownian excursion distance, δx. Collagen length change is measuredas follows. Since the refractive index of the quantum dot and theexperimental media are different, the point spread function (PSF) of thequantum dots varies based on depth in the media. By using a model of thepredicted point spread function developed by Aquet et al. (Confocal,Multiphoton, and Nonlinear Microscope Imaging II (2005)), the z-positionis determined with precision in the nanometer range by fitting theactual PSF to the model. A 60× objective is focused onto the collagentethered SPM beads with the quantum dots glass-bound position out offocus. The quantum dot positions are determined with greater accuracyusing defocused particles. Bead position relative to the focal plane isestimated by fitting a circle to the outline of the bead, and the PSFfit determines the position of the quantum dot. This distance ismonitored for changes as enzyme is introduced into the chamber. Brownianmotion changes are measured as follows. Since the catabolic enzymes bindtightly to the collagen molecule (stiffening) and comprise a significantpercentage of collagen's molecular weight (33% for BC), binding altersthe Brownian excursion. Thus, x-y position is monitored following theintroduction of enzyme to the chamber.

A high-resolution camera (Retiga EXL, QI Imaging, 1394b, Mono, 14 bitcooled OC) is added to a Nikon TE2000E microscope, which enablesvertical, spatial and temporal resolution to a level that allowsdetection of changes in the relative position of particles on the orderof nanometers. Bead and quantum dot positions are reported as functionsof time, and the relative displacement curves are examined for abruptchanges.

Results

Strain applied to collagen monomers modifies the binding ratecoefficient and the cleavage rate coefficient. Effective cleavage ratesof collagen by enzymes are the result of a binding and cleavage event.Both of these events involve physical interaction of the enzyme withspecific sites on the collagen monomer and are affected by theapplication of strain. The detection of the binding events allows adirect determination of the extent each event (binding or cleavage) isinfluenced by mechanical strain.

C. Effect of Mechanical Force on the Physical Behavior of CollagenMolecular Structure and on Hierarchical Collagen Assemblies

Methods

Atomistic-based multi-scale modeling is used for the chemomechanicalbehavior of tropocollagen monomers and the hierarchical structure ofcollagen fibrils, focusing on the effect of strain, enzymatic activity,and the coupling between strain and cleavage probability. The mechanicalproperties of tropocollagen molecules are investigated using steeredmolecular dynamics simulations using both a CHARMM force field as wellas the new first principles based reactive force field ReaxFF. TheReaxFF model includes a first principles based description of allchemical bonds in the system (including breaking and formation ofcovalent bonds). This method captures chemomechanical coupling. TheReaxFF model bridges the scale from first principles quantum mechanicaldescriptions (limited to <200 atoms) to empirical CHARMM-like forcefields and molecular mechanics models. In ReaxFF, the energy of atomsand molecules and their charges is not calculated based on simpledistance-energy relations, but instead, is calculated dependent on thequantum mechanical state or bond orders.

The software THeBuScr (Triple-Helical collagen Building Script) is usedto build a model of the tropocollagen molecule. This builder enables thegeneration of tropocollagen monomers with any desired sequence. Thesequence is chosen based on those structures relevant to the cleavagedomain in the collagen molecule. The MMP enzyme structures are takenfrom the protein data bank (PDB).

The tropocollagen molecule is subjected to traction along its principalaxis using steered molecular dynamics (SMD). Other studies are focusedon coupling between mechanical strain and the effect on the collagennetwork formation and stability. The interaction of a singletropocollagen molecule with MMP is studied under varying stain. Theenergy landscape of these atomic mechanisms is mapped and quantitativeinformation is provided of how strain affects the interaction of theenzyme with the collagen. This series of computational experimentsdiscerns that binding affinity and/or cleavage ability (modeled as thechange in energy used to separate an alpha chain from the triple helix)is affected by the applied load. Using a combination with mesoscalesimulations, entropic effects are captured that may play a key role atlonger time-scales and, in the case of longer tropocollagen monomersections, during assembly processes. The interaction and assembly of alarge ensemble of >10,000 tropocollagen monomers is simulated undervarying mechanical strain and under varying enzymatic activity.

Example 13 Producing Organized Collagen by Strain-Stabilization Effect

Collagen fibrils are protected from enzymatic attack if they are“strained” appropriately. The methods described in Example 2 aremodified by applying a strain field to the construct, creating a strainon the order of 1.5% to 4% where fibril retention is desired. Fibrilsoutside this range are preferentially degraded. Controllable strain iscreated by fixing the organized collagen gels to moveable grips orfunctionalized surfaces (see, e.g., Ruberti et al., Biochem. Biophys.Res. Commun. 336:483-489 (2005)). FIG. 25 shows two devices that areused to apply known loads to collagen constructs. Both devices allowhigh-powered optical observation and media access for enzyme and monomercycling. These devices create uniaxial and tangential loads suitable forthe production tendon-like and cornea-like organization.

EQUIVALENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of producing an organized array of collagen fibrils, themethod comprising: contacting a template with a still solutioncomprising collagen monomers in liquid crystalline phase; andneutralizing the solution in contact with the template, thereby inducingthe assembly of the collagen monomers into an organized array ofcollagen fibrils.
 2. The method of claim 1, wherein the collagenmonomers comprise a nematic phase.
 3. The method of claim 1, wherein thecollagen monomers comprise a smectic phase.
 4. The method of claim 1,wherein the collagen monomers comprise a cholesteric phase.
 5. Themethod of claim 1, wherein the solution comprises about 30 mg/ml toabout 500 mg/ml collagen monomers.
 6. The method of claim 1, wherein theneutralizing step comprises adjusting the solution to a pH of about 5 toabout
 10. 7. The method of claim 6, further comprising neutralizing thesolution in contact with the template at about 10° C. to about 39° C. 8.The method of claim 1, wherein the template comprises one or moreguidance structures.
 9. The method of claim 8, wherein the one or moreguidance structures are one or more internal guidance structures and thetemplate is placed in a stationary position within the solution.
 10. Themethod of claim 9, wherein the one or more internal guidance structurescomprise a high aspect ratio geometry.
 11. The method of claim 10,wherein the one or more internal guidance structures comprise a minorlength scale of between about 14 nm and about 20 μm.
 12. The method ofclaim 9, wherein the one or more internal guidance structures comprise abiodegradable material.
 13. The method of claim 1, wherein the templatecomprises a plurality of external guidance structures having aninterstructure distance of about 2 μm to about 200 μm.
 14. The method ofclaim 1, wherein the template comprises a cylindrical tube, twoconcentric cylindrical tubes, or two concentric hemispheres.
 15. Themethod of claim 14, wherein the template comprises a cylindrical tubehaving an inner diameter of about 100 μm to about 1 mm.
 16. The methodof claim 14, wherein the template comprises two concentric cylinderswith a gap width of about 2 μm to about 4 mm.
 17. The method of claim14, wherein the template comprises two concentric hemispheres with a gapwidth of about 2 μm to about 4 mm.
 18. The method of claim 1, whereinthe template comprises one or more internal guidance structures and oneor more external guidance structures.
 19. The method of claim 1, whereinthe solution comprises one or more co-nonsolvency agents.
 20. The methodof claim 1, wherein the solution further comprises a collagen bindingagent.
 21. The method of claim 1, further comprising applying anelectric charge to the contacted template.
 22. The method of claim 1,wherein the organized array of collagen fibrils is about 100 μm to about30 cm in length.
 23. The method of claim 1, wherein the organized arrayof collagen fibrils comprises D-banded collagen fibrils.
 24. The methodof claim 1, further comprising contacting the collagen monomers in theorganized array of collagen fibrils with a crosslinking agent.
 25. Themethod of claim 8, further comprising modulating the surface energy ofthe guidance structures.
 26. The method of claim 8, wherein the guidancestructures comprise a surface having a pattern of hydrophobic andhydrophilic stripes.
 27. The method of claim 1, further comprisingorganizing the array of collagen fibrils into a tissue.
 28. An organizedarray of collagen fibrils produced by the method of claim
 1. 29. Amethod of directing the assembly of collagen fibrils, the methodcomprising: contacting a template with a still solution comprisingcollagen monomers in liquid crystalline phase, the template comprising aplurality of guidance structures; and neutralizing the solution incontact with the template, wherein contacting the template directs theassembly of the collagen monomers in a pattern or direction defined bythe guidance structures.